Isolated gene for methylmalonyl CoA epimerase and uses thereof

ABSTRACT

Recombinant host cells that comprise recombinant DNA expression vectors that drive expression of a product and a precursor for biosynthesis of that product can be used to produce useful products such as polyketides in host cells that do not naturally produce the product or produce the product or precursor at low levels due to the absence of the precursor or the presence of the precursor in rate limiting amounts.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U.S. patentapplication Ser. No. 60/161,703, filed Oct. 27, 1999, and is related toSer. No. 60/161,414, filed Oct. 25, 1999, and 60/206,082, filed May 18,2000, each of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention provides recombinant methods and materialsfor producing polyketides by recombinant DNA technology. The inventionrelates to the fields of agriculture, animal husbandry, chemistry,medicinal chemistry, medicine, molecular biology, pharmacology, andveterinary technology.

BACKGROUND OF THE INVENTION

[0003] Polyketides represent a large family of diverse compoundssynthesized from 2-carbon units through a series of condensations andsubsequent modifications. Polyketides occur in many types of organisms,including fungi and mycelial bacteria, in particular, the actinomycetes.There are a wide variety of polyketide structures, and the class ofpolyketides encompasses numerous compounds with diverse activities.Erythromycin, FK-506, FK-520, megalomicin, narbomycin, oleandomycin,picromycin, rapamycin, spinocyn, and tylosin are examples of suchcompounds. Given the difficulty in producing polyketide compounds bytraditional chemical methodology, and the typically low production ofpolyketides in wild-type cells, there has been considerable interest infinding improved or alternate means to produce polyketide compounds. SeePCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; 97/02358;and 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639;5,672,491; 5,712,146; and 5,962,290; and Fu et al., 1994, Biochemistry33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr,1995, Angew. Chem. Int. Ed. Engl. 34(8): 881-888, each of which isincorporated herein by reference.

[0004] Polyketides are synthesized in nature by polyketide synthase(PKS) enzymes. These enzymes, which are complexes of multiple largeproteins, are similar to the synthases that catalyze condensation of2-carbon units in the biosynthesis of fatty acids. PKS enzymes areencoded by PKS genes that usually consist of three or more open readingframes (ORFs). Two major types of PKS enzymes are known; these differ intheir composition and mode of synthesis. These two major types of PKSenzymes are commonly referred to as Type I or “modular” and Type II“iterative” PKS enzymes. A third type of PKS found primarily in fungalcells has features of both the Type I and Type II enzymes and isreferred to as a “fungal” PKS enzymes.

[0005] Modular PKSs are responsible for producing a large number of 12-,14-, and 16-membered macrolide antibiotics including erythromycin,megalomicin, methymycin, narbomycin, oleandomycin, picromycin, andtylosin. Each ORF of a modular PKS can comprise one, two, or more“modules” of ketosynthase activity, each module of which consists of atleast two (if a loading module) and more typically three (for thesimplest extender module) or more enzymatic activities or “domains.”These large multifunctional enzymes (>300,000 kDa) catalyze thebiosynthesis of polyketide macrolactones through multistep pathwaysinvolving decarboxylative condensations between acyl thioesters followedby cycles of varying β-carbon processing activities (see O'Hagan, D. Thepolyketide metabolites; E. Horwood: New York, 1991, incorporated hereinby reference).

[0006] During the past half decade, the study of modular PKS functionand specificity has been greatly facilitated by the plasmid-basedStreptomyces coelicolor expression system developed with the6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al.,1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262:1546-1557, and U.S. Pat. Nos. 5,672,491 and 5,712,146, each of which isincorporated herein by reference). The advantages to this plasmid-basedgenetic system for DEBS are that it overcomes the tedious and limitedtechniques for manipulating the natural DEBS host organism,Saccharopolyspora erythraea, allows more facile construction ofrecombinant PKSs, and reduces the complexity of PKS analysis byproviding a “clean” host background. This system also expeditedconstruction of the first combinatorial modular polyketide library inStreptomyces (see PCT publication Nos. WO 98/49315 and 00/024907, eachof which is incorporated herein by reference).

[0007] The ability to control aspects of polyketide biosynthesis, suchas monomer selection and degree of β-carbon processing, by geneticmanipulation of PKSs has stimulated great interest in the combinatorialengineering of novel antibiotics (see Hutchinson, 1998, Curr. Opin.Microbiol. 1: 319-329; Carreras and Santi, 1998, Curr. Opin. Biotech. 9:403-411; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which isincorporated herein by reference). This interest has resulted in thecloning, analysis, and manipulation by recombinant DNA technology ofgenes that encode PKS enzymes. The resulting technology allows one tomanipulate a known PKS gene cluster either to produce the polyketidesynthesized by that PKS at higher levels than occur in nature or inhosts that otherwise do not produce the polyketide. The technology alsoallows one to produce molecules that are structurally related to, butdistinct from, the polyketides produced from known PKS gene clusters.

[0008] There has been a great deal of interest in expressing polyketidesproduced by Type I and Type II PKS enzymes in host cells that do notnormally express such enzymes. For example, the production of the fungalpolyketide 6-methylsalicylic acid (6-MSA) in heterologous E. coli,yeast, and plant cells has been reported. See Kealey et al., January1998, Production of a polyketide natural product innonpolyketide-producing prokaryotic and eukaryotic host, Proc. Natl.Acad. Sci. USA 95:505-9, U.S. Pat. No. 6,033,883, and PCT PatentPublication Nos. 98/27203 and 99/02669, each of which is incorporatedherein by reference. Heterologous production of 6-MSA required or wasconsiderably increased by co-expression of a heterologous acyl carrierprotein synthase (ACPS) and that, for E. coli, media supplements werehelpful in increasing the level of the malonyl CoA substrate utilized in6-MSA biosynthesis. See also, PCT Patent Publication No. 97/13845,incorporated herein by reference.

[0009] The biosynthesis of other polyketides requires substrates otherthan or in addition to malonyl CoA. Such substrates include, forexample, propionyl CoA, 2-methylmalonyl CoA, 2-hydroxymalonyl CoA, and2-ethylmalonyl CoA. Of the myriad host cells possible for utilization aspolyketide producing hosts, many do not naturally produce suchsubstrates. Given the potential for making valuable and usefulpolyketides in large quantities in heterologous host cells, there is aneed for host cells capable of making the substrates required forpolyketide biosynthesis. The present invention helps meet that need byproviding recombinant host cells, expression vectors, and methods formaking polyketides in diverse host cells.

SUMMARY OF THE INVENTION

[0010] The present invention provides recombinant host cells andexpression vectors for making products in host cells that are otherwiseunable to make those products due to the lack of a biosynthetic pathwayto produce a precursor required for biosynthesis of the product. Thepresent invention also provides methods for increasing the amounts of aproduct produced in a host cell by providing recombinant biosyntheticpathways for production of a precursor utilized in the biosynthesis of aproduct.

[0011] In one embodiment, the host cell does not produce the precursor,and the host cell is modified by introduction of a recombinantexpression vector so that it can produce the precursor. In anotherembodiment, the precursor is produced in the host cell in small amounts,and the host cell is modified by introduction of a recombinantexpression vector so that it can produce the precursor in largeramounts. In a preferred embodiment, the precursor is a primarymetabolite that is produced in first cell but not in a secondheterologous cell. In accordance with the methods of the invention, thegenes that encode the enzymes that produce the primary metabolite in thefirst cell are transferred to the second cell. The transfer isaccomplished using an expression vector of the invention. The expressionvector drives expression of the genes and production of the metabolitein the second cell.

[0012] In a preferred embodiment, the product is a polyketide. Thepolyketide is a polyketide synthesized by either a modular, iterative,or fungal PKS. The precursor is selected from the group consisting ofmalonyl CoA, propionyl CoA, methylmalonyl CoA, ethylmalonyl CoA, andhydroxymalonyl or methoxymalonyl CoA. In an especially preferredembodiment, the polyketide utilizes methylmalonyl CoA in itsbiosynthesis. In one preferred embodiment, the polyketide is synthesizedby a modular PKS that requires methylmalonyl CoA to synthesize thepolyketide.

[0013] In one embodiment, the host cell is either a procaryotic oreukaryotic host cell. In one embodiment, the host cell is an E. colihost cell. In another embodiment, the host cell is a yeast host cell. Inanother embodiment, the host cell is an Actinomycetes host cell,including but not limited to a Streptomyces host cell. In anotherembodiment, the host cell is a plant host cell. In a preferredembodiment, the host cell is either an E. coli or yeast host cell, theproduct is a polyketide, and the precursor is methylmalonyl CoA.

[0014] In one embodiment, the invention provides a recombinantexpression vector that comprises a promoter positioned to driveexpression of one or more genes that encode the enzymes required forbiosynthesis of a precursor. In a preferred embodiment, the promoter isderived from a PKS gene. In a related embodiment, the invention providesrecombinant host cells comprising one or more expression vectors thatdrive expression of the enzymes that produce the precursor.

[0015] In another embodiment, the invention provides a recombinant hostcell that comprises not only an expression vector of the invention butalso an expression vector that comprises a promoter positioned to driveexpression of a PKS. In a related embodiment, the invention providesrecombinant host cells comprising the vector that produces the PKS andits corresponding polyketide. In a preferred embodiment, the host cellis an E. coli or yeast host cell.

[0016] These and other embodiments of the invention are described inmore detail in the following description, the examples, and claims setforth below.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1 shows the modules and domains of DEBS and the biosynthesisof 6-dEB from propionyl CoA and methylmalonyl CoA.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides recombinant host cells andexpression vectors for making products in host cells, which areotherwise unable to make those products due to the lack of abiosynthetic pathway to produce a precursor required for biosynthesis ofthe product. As used herein, the term recombinant refers to a cell,compound, or composition produced at least in part by humanintervention, particularly by modification of the genetic material. Thepresent invention also provides methods for increasing the amounts of aproduct produced in a host cell by providing recombinant biosyntheticpathways for production of a precursor utilized in the biosynthesis of aproduct.

[0019] In one embodiment, the host cell does not produce the precursor,and the host cell is modified by introduction of a recombinantexpression vector so that it can produce the precursor. In anotherembodiment, the precursor is produced in the host cell in small amounts,and the host cell is modified by introduction of a recombinantexpression vector so that it can produce the precursor in largeramounts. In a preferred embodiment, the precursor is a primarymetabolite that is produced in first cell but not in a secondheterologous cell. In accordance with the methods of the invention, thegenes that encode the enzymes that produce the primary metabolite in thefirst cell are transferred to the second cell. The transfer isaccomplished using an expression vector of the invention. The expressionvector drives expression of the genes and production of the metabolitein the second cell.

[0020] The invention, in its most general form, concerns theintroduction, in whole or in part, of a metabolic pathway from one cellinto a heterologous host cell. The invention also encompasses themodification of an existing metabolic pathway, in whole or in part, in acell, through the introduction of heterologous genetic material into thecell. In all embodiments, the resulting cell is different with regard toits cellular physiology and biochemistry in a manner such that thebio-synthesis, bio-degradation, transport, biochemical modification, orlevels of intracellular metabolites allow production or improveexpression of desired products. The invention is exemplified byincreasing the level of polyketides produced in a heterologous host andby restricting the chemical composition of products to the desiredstructures.

[0021] Thus, in a preferred embodiment, the product produced by the cellis a polyketide. The polyketide is a polyketide synthesized by either amodular, iterative, or fungal PKS. The precursor is selected from thegroup consisting of malonyl CoA, propionyl CoA, methylmalonyl CoA,ethylmalonyl CoA, and hydroxymalonyl or methoxymalonyl CoA. In anespecially preferred embodiment, the polyketide utilizes methylmalonylCoA in its biosynthesis. In one preferred embodiment, the polyketide issynthesized by a modular PKS that requires methylmalonyl CoA tosynthesize the polyketide.

[0022] The polyketide class of natural products includes members havingdiverse structural and pharmacological properties (see Monaghan andTkacz, 1990, Annu. Rev. Microbiol. 44: 271, incorporated herein byreference). Polyketides are assembled by polyketide synthases throughsuccessive condensations of activated coenzyme-A thioester monomersderived from small organic acids such as acetate, propionate, andbutyrate. Active sites required for condensation include anacyltransferase (AT), acyl carrier protein (ACP), andbeta-ketoacylsynthase (KS). Each condensation cycle results in a β-ketogroup that undergoes all, some, or none of a series of processingactivities. Active sites that perform these reactions include aketoreductase (KR), dehydratase (DH), and enoylreductase (ER). Thus, theabsence of any beta-keto processing domain results in the presence of aketone, a KR alone gives rise to a hydroxyl, a KR and DH result in analkene, while a KR, DH, and ER combination leads to complete reductionto an alkane. After assembly of the polyketide chain, the moleculetypically undergoes cyclization(s) and post-PKS modification (e.g.glycosylation, oxidation, acylation) to achieve the final activecompound.

[0023] Macrolides such as erythromycin and megalomicin are synthesizedby modular PKSs (see Cane et al., 1998, Science 282: 63, incorporatedherein by reference). For illustrative purposes, the PKS that producesthe erythromycin polyketide (6-deoxyerythronolide B synthase or DEBS;see U.S. Pat. No. 5,824,513, incorporated herein by reference) is shownin FIG. 1. DEBS is the most characterized and extensively used modularPKS system. DEBS synthesizes the polyketide 6-deoxyerythronolide B(6-dEB) from propionyl CoA and methylmalonyl CoA. In modular PKS enzymessuch as DEBS, the enzymatic steps for each round of condensation andreduction are encoded within a single “module” of the polypeptide (i.e.,one distinct module for every condensation cycle). DEBS consists of aloading module and 6 extender modules and a chain terminatingthioesterase (TE) domain within three extremely large polypeptidesencoded by three open reading frames (ORFs, designated eryAI, eryAII,and eryAIII).

[0024] Each of the three polypeptide subunits of DEBS (DEBSI, DEBSII,and DEBSIII) contains 2 extender modules, DEBS additionaly contains theloading module. Collectively, these proteins catalyze the condensationand appropriate reduction of 1 propionyl CoA starter unit and 6methylmalonyl CoA extender units. Modules 1, 2, 5, and 6 contain KRdomains; module 4 contains a complete set, KR/DH/ER, of reductive anddehydratase domains; and module 3 contains no functional reductivedomain. Following the condensation and appropriate dehydration andreduction reactions, the enzyme bound intermediate is lactonized by theTE at the end of extender module 6 to form 6-dEB.

[0025] More particularly, the loading module of DEBS consists of twodomains, an acyl-transferase (AT) domain and an acyl carrier protein(ACP) domain. In other PKS enzymes, the loading module is not composedof an AT and an ACP but instead utilizes a partially inactivated KS, anAT, and an ACP. This partially inactivated KS is in most instancescalled KSQ, where the superscript letter is the abbreviation for theamino acid, glutamine, that is present instead of the active sitecysteine required for full activity. The AT domain of the loading modulerecognizes a particular acyl CoA (propionyl for DEBS, which can alsoaccept acetyl) and transfers it as a thiol ester to the ACP of theloading module. Concurrently, the AT on each of the extender modulesrecognizes a particular extender-CoA (methylmalonyl for DEBS) andtransfers it to the ACP of that module to form a thioester. Once the PKSis primed with acyl- and malonyl-ACPs, the acyl group of the loadingmodule migrates to form a thiol ester (trans-esterification) at the KSof the first extender module; at this stage, extender module 1 possessesan acyl-KS and a methylmalonyl ACP. The acyl group derived from theloading module is then covalently attached to the alpha-carbon of themalonyl group to form a carbon-carbon bond, driven by concomitantdecarboxylation, and generating a new acyl-ACP that has a backbone twocarbons longer than the loading unit (elongation or extension). Thegrowing polyketide chain is transferred from the ACP to the KS of thenext module, and the process continues.

[0026] The polyketide chain, growing by two carbons each module, issequentially passed as a covalently bound thiol ester from module tomodule, in an assembly line-like process. The carbon chain produced bythis process alone would possess a ketone at every other carbon atom,producing a poyketone, from which the name polyketide arises. Commonly,however, the beta keto group of each two-carbon unit is modified justafter it has been added to the growing polyketide chain but before it istransferred to the next module by either a KR, a KR plus a DH, or a KR,a DH, and an ER. As noted above, modules may contain additionalenzymatic activities as well.

[0027] Once a polyketide chain traverses the final extender module of aPKS, it encounters the releasing domain or thioesterase found at thecarboxyl end of most PKSs. Here, the polyketide is cleaved from theenzyme and typically cyclyzed. The resulting polyketide can be modifiedfurther by tailoring or modification enzymes; these enzymes addcarbohydrate groups or methyl groups, or make other modifications, i.e.,oxidation or reduction, on the polyketide core molecule. For example,the final steps in conversion of 6-dEB to erythromycin A include theactions of a number of modification enzymes, such as: C-6 hydroxylation,attachment of mycarose and desosamine sugars, C-12 hydroxylation (whichproduces erythromycin C), and conversion of mycarose to cladinose viaO-methylation.

[0028] With this overview of PKS and post-PKS modification enzymes andtheir substrates, one can better appreciate the benefits provided by thepresent invention. DEBS is produced naturally in Saccharopolysporaerythraea and has been transferred to a variety of Streptomyces species,such as S. coelicolor CH999 and S. lividans K4-114 and K4-155, in whichit functions without further modification of the host cell to produce6-dEB. Thus, S. erythraea, S. coelicolor, and S. lividans make therequired precursors for 6-dEB synthesis. However, many othernon-Saccharopolyspora, non-Streptomyces host cells do not make all ofthe required precursors or make them only at levels sufficient tosupport only very small amounts of polyketide biosynthesis.

[0029] The present invention provides recombinant DNA expression vectorsand methods for making a polyketide and its required precursors in anyhost cell. In one embodiment, the host cell is either a procaryotic oreukaryotic host cell. In a preferred embodiment, the host cell is an E.coli host cell. In another preferred embodiment, the host cell is ayeast host cell. In another embodiment, the host cell is a plant hostcell. In a preferred embodiment, the host cell is either an E. coli oryeast host cell, the product is a polyketide, and the precursor ismethylmalonyl CoA.

[0030] The recombinant expression vectors of the invention comprise apromoter positioned to drive expression of one or more genes that encodethe enzymes required for biosynthesis of a precursor. In a preferredembodiment, the promoter is derived from a PKS gene. In anotherpreferred embodiment, the promoter is one derived from a host cell geneor from a virus or phage that normally infects the host cell and isheterologous to the gene that encodes the biosynthetic enzyme.

[0031] In another embodiment, the invention provides a recombinant hostcell that comprises not only an expression vector of the invention butalso an expression vector that comprises a promoter positioned to driveexpression of a PKS. In a related embodiment, the invention providesrecombinant host cells comprising the vector that produces the PKS andits corresponding polyketide. In a preferred embodiment, the host cellis an E. coli or yeast host cell.

[0032] Neither E. coli nor yeast makes sufficient methylmalonyl CoA tosupport biosynthesis of large amounts of polyketides that requiremethylmalonyl CoA in their biosynthesis, and most species do not producethe methylmalonyl CoA substrate at all. In one embodiment, the presentinvention provides E. coli, yeast, and other host cells that producemethylmalonyl CoA in amounts sufficient to support polyketidebiosynthesis. In preferred embodiments, the cells produce sufficientamounts of methylmalonyl CoA to support biosynthesis of polyketidesrequiring methylmalonyl CoA for their biosynthesis at levels rangingfrom 1 μg/L, to 1 mg/L, to 10 mg/L, to 100 mg/L, to 1 g/L, to 10 g/L.

[0033] In one embodiment, the host cells of the invention have beenmodified to express a heterologous methylmalonyl CoA mutase. Thisenzyme, which converts succinyl CoA to methylmalonyl CoA (although thereverse reaction is 20 times more favored) has been expressed in E. coliusing a gene cloned from propionibacteria but was inactive due to thelack of vitamin B12. In accordance with the methods of the presentinvention, this enzyme can be made in an active form in E. coli andother host cells by either expressing (constitutively or otherwise) aB12 transporter gene, such as the endogenous E. coli gene and/or byutilizing a media that facilitates B12 uptake (as used herein, B12 canrefer to the precursor hydroxocobalamin, which is converted to B12).While certain methylmalonyl CoA mutases make the R-isomer, including themethylmalonyl CoA mutases derived from the propionibacteria, theR-isomer can be converted to the S-isomer using an epimerase. Forexample, epimerase genes from propionibacteria or Streptomyces can beemployed for this purpose.

[0034] In another embodiment, the host cells of the invention have beenmodified to express a heterologous propionyl CoA carboxylase thatconverts propionyl CoA to methylmalonyl CoA. In this embodiment, one canfurther increase the amount of methylmalonyl CoA precursor by culturingthe cells in a media supplemented with propionate. In a preferredembodiment, the host cells are E. coli host cells.

[0035] Thus, in accordance with the methods of the invention, theheterologous production of certain polyketides in E. coli, yeast, andother host organisms require both the heterologous expression of adesired PKS and also the enzymes that produce at least some of thesubstrate molecules required by the PKS. These substrate molecules,called precursors, are not normally found as intracelluar metabolites inthe host organism or are present in low abundance. The present inventionprovides a method to produce or modify the composition or quantities ofintracellular metabolites within a host organism where such metabolitesare not naturally present or are present in non-optimal amounts.

[0036] A specific embodiment of the present invention concerns theintroduction and modification of biochemical pathways for methylmalonylCoA biosynthesis. Methylmalonyl CoA, as noted above, is a substrateutilized for the synthesis of polyketides by many polyketide synthases.Some of the known biochemical pathways for the intracellular productionof methylmalonyl CoA employ enzymes and their corresponding genes foundin certain organisms. These enzymes and genes have not been found, orare otherwise non-optimal, in other organisms. These other organismsinclude those that could otherwise be very useful as heterologous hostsfor the production of polyketides. The present invention providesmethods to engineer a host organism so that it contains a new ormodified ability to produce methylmalonyl CoA and/or to increase ordecrease the levels of methylmalonyl CoA in the host.

[0037] As noted above, two biochemical pathways involving methylmalonylCoA are particularly relevant to this aspect of the present invention.These pathways are the methylmalonyl CoA mutase pathway, hereafterreferred to as the MUT pathway, and the propionyl CoA carboxylasepathway, hereafter referred to as the PCC pathway.

[0038] The MUT pathway includes the enzymes methylmalonyl CoA mutase(5.4.99.2, using the numbering system devised by the NomenclatureCommittee of the International Union of biochemistry and MolecularBiology), methylmalonyl CoA epimerase (5.1.99.1), and malonyl CoAdecarboxylase (4.1.1.9). The biochemical pathway includes the conversionof succinyl CoA to (R)-methylmalonyl CoA through the action ofmethylmalonyl CoA mutase (5.4.99.2) followed by the conversion of(R)-methylmalonyl CoA to (S)-methylmalonyl CoA through the action ofmethylmalonyl CoA epimerase (5.1.99.1). (S)-methylmalonyl CoA is asubstrate utilized by several polyketide synthases. The enzyme malonylCoA decarboxylase (4.1.1.9) catalyzes the decarboxylation of malonyl CoAbut is also reported to catalyze the decarboxylation of(R)-methylmalonyl CoA to form propionyl CoA. Propionyl CoA is asubstrate utilized by some polyketide synthases.

[0039] The PCC pathway includes the enzymes propionyl CoA carboxylase(6.4.1.3) and propionyl CoA synthetase (6.2.1.17). The biochemicalpathway includes the conversion of propionate to propionyl CoA throughthe action of propionyl CoA synthetase (6.2.1.17) followed by theconversion of propionyl CoA to (S)-methylmalonyl CoA through the actionof propionyl CoA carboxylase (6.4.1.3). (S)-methylmalonyl CoA is thesubstrate utilized by many polyketide synthases.

[0040] An illustrative embodiment of the present invention employsspecific enzymes from these pathways. As those skilled in the art willrecognize upon contemplation of this description of the invention, theinvention can also be practiced using additional and/or alternativeenzymes involved in the MUT and PCC pathways. Moreover, the inventioncan be practiced using additional and alternative pathways formethylmalonyl CoA and other intracelluar metabolites.

[0041] The methods of the invention involve the introduction of geneticmaterial into a host strain of choice to modify or alter the cellularphysiology and biochemistry of the host. Through the introduction ofgenetic material, the host strain acquires new properties, e.g. theability to produce a new, or greater quantities of, an intracellularmetabolite. In an illustrative embodiment of the invention, theintroduction of genetic material into the host strain results in a newor modified ability to produce methylmalonyl CoA. The genetic materialintroduced into the host strain contains gene(s), or parts of genes,coding for one or more of the enzymes involved in thebio-synthesis/bio-degradation of methylmalonyl CoA and may also includeadditional elements for the expression and/or regulation of expressionof these genes, e.g. promoter sequences. Specific gene sequences codingfor enzymes involved in the bio-synthesis/bio-degradation ofmethylmalonyl CoA are listed below.

[0042] A suitable methylmalonyl CoA mutase (5.4.99.2) gene can beisolated from Streptomyces cinnamonensis. See Birch et al., 1993, J.Bacteriol. 175: 3511-3519, entitled “Cloning, sequencing, and expressionof the gene encoding methylmalonyl-coenzyme A mutase from Streptomycescinnamonensis.” This enzyme is a two subunit enzyme; the A and B subunitcoding sequences are available under Genbank accession L10064. Anothersuitable methylmalonyl CoA mutase gene can be isolated fromPropionibacterium shermanii. See Marsh et al., 1989, Biochem. J. 260:345-352, entitled “Cloning and structural characterization of the genescoding for adenosylcobalamin-dependent methylmalonyl CoA mutase fromPropionibacterium shermanii.” Alternatively, a suitable methylmalonylCoA mutase gene can be isolated from Porphyromonas gingivalis. SeeJackson et al., 1995, Gene 167: 127-132, entitled “Cloning, expressionand sequence analysis of the genes encoding the heterodimericmethylmalonyl CoA mutase of Porphyromonas gingivalis W50.”Alternatively, suitable methylmalonyl CoA mutase genes can be isolatedfrom any of the sources noted in the following table of a partial BLASTsearch report or from additional BLAST analyses.

Results of BLAST Search of NCBI Database for Methylmalonyl CoA MutaseMutA

[0043] gb|L10064|STMMUTA Streptomyces cinnamonensis 931 0.0 (querysequence)

[0044] gb|AD000015|MSGY175 Mycobacterium tuberculosis sequence 300 7e-80

[0045] emb|Z79701|MTCY277 Mycobacterium tuberculosis H37Rv 300 7e-80

[0046] gb|AD000001|MSGY456 Mycobacterium tuberculosis sequence 238 8e-76

[0047] emb|X14965|PSMUTAB Propionibacterium shermanii mutA 268 5e-70

[0048] gb|L30136|POYMCMAB Porphyromonas gingivalis 137 9e-31

[0049] gb|AE000375|AE000375 Escherichia coli K-12 MG1655 134 1e-29

[0050] gb|U28377|ECU28377 Escherichia coli K-12 genome; 134 1e-29

[0051] emb|X66836|ECSERAICI E.coli serA, iciA, sbm genes 133 1e-29

[0052] gb|AF080073|SMPCAS2 Sinorhizobium meliloti 130 2e-28

[0053] ref|NM_(—)000255.1|MUT| Homo sapiens 113 2e-23

[0054] dbj|AP000006|AP000006 Pyrococcus horikoshii OT3 110 2e-22

[0055] emb|AJ248285.1|CNSPAX03 Pyrococcus abyssi 109 3e-22

[0056] emb|X51941|MMMMCOAM Mouse mRNA 109 3e-22

[0057] gb|AE000952|AE000952 Archaeoglobus fulgidus section 155 104 9e-21

[0058] emb|AJ237976.1|SCO237976 Streptomyces coelicolor icmA gene 1032e-20

[0059] dbj|AP000062.1|AP000062 Aeropyrum pernix genomic DNA 102 3e-20

[0060] gb|U67612|SCU67612 Streptomyces cinnamonensis coenzyme B12 987e-19

[0061] gb|AE001015|AE001015 Archaeoglobus fulgidus section 92 97 1e-18

[0062] emb|X59424|BFOF4 Bacillus firmus OF4 genes for ATP binding 827e-14

MutB

[0063] gb|L10064|STMMUTA Streptomyces cinnamonensis 1379 0.0 (querysequence)

[0064] gb|AD000001|MSGY456 Mycobacterium tuberculosis 1018 0.0

[0065] emb|Z79701|MTCY277 Mycobacterium tuberculosis H37Rv 1017 0.0

[0066] gb|AD000015|MSGY175 Mycobacterium tuberculosis sequence 1017 0.0

[0067] emb|X14965|PSMUTAB Propionibacterium shermanii 996 0.0

[0068] gb|L30136|POYMCMAB Porphyromonas gingivalis methylmalonyl 882 0.0

[0069] ref|NM_(—)000255.1|MUT| Homo sapiens methylmalonyl Coenzyme A 8550.0

[0070] emb|X51941|MMMMCOAM Mouse mRNA 32 0.0

[0071] gb|U28377|ECU28377 Escherichia coli K-12 genome 798 0.0

[0072] gb AE000375|AE000375 Escherichia coli K-12 MG1655 798 0.0

[0073] emb|X66836|ECSERAICI E.coli serA, iciA, sbm genes 797 0.0

[0074] gb|AF080073|SMPCAS2 Sinorhizobium meliloti 782 0.0

[0075] gb|AE001015|AE001015 Archaeoglobus fulgidus 516 e-145

[0076] dbj|AP000062.1|AP000062 Aeropyrum pernix genonic DNA 408 e-139

[0077] emb|AJ248285.1|CNSPAX03 Pyrococcus abyssi complete genome 486e-135

[0078] dbj|AP000006|AP000006 Pyrococcus horikoshii OT3 genomic DNA 480e-133

[0079] gb|AE000952|AE000952 Archaeoglobus fulgidus section 155 467 e-130

[0080] emb|Z35604.1|CEZK1058 Caenorhabditis elegans cosmid ZK1058 316e-109

[0081] emb|AJ237976.1|SCO237976 Streptomyces coelicolor icmA 377 e-103

[0082] gb|U67612|SCU67612 Streptomyces cinnamonensis coenzyme 372 e-101

[0083] emb|AL035161|SC9C7 Streptomyces coelicolor cosmnid 9C7 359 2e-97

[0084] gb|U28335|MEU28335 Methylobacterium extorquens 351 4e-95

[0085] gb|AF008569|AF008569 Streptomyces collinus coenzyme 337 8e-91

[0086] gb|U65074|ECU65074 Escherichia coli chromosome 275 3e-72

[0087] gb|M37500|HUMMUT03 Human methylmalonyl CoA mutase 202 3e-50

[0088] gb|AF178673.1|AF178673 Streptomyces cinnamonensis 183 1e-44

[0089] emb|Z49936.1|CEF13B10 Caenorhabditis elegans cosmid F13B10 1382e-41

[0090] gb|M37499|HUMMUT02 Human methylmalonyl CoA mutase 112 4e-23

[0091] dbj|AP000001.1|AP000001 Pyrococcus horikoshii OT3 genomic 1062e-21

[0092] emb|AJ248283.1|CNSPAX01 Pyrococcus abyssi complete genome 1062e-21

[0093] gb|M37503|HUMMUT06 Human methylmalonyl CoA mutase 101 7e-20

[0094] gb|M37508|HUMMUT11 Human methylmalonyl CoA mutase 86 3e-15

[0095] gb|M37509|HUMMUT12 Human methylmalonyl CoA mutase 80 3e-13

[0096] gb|M37501|HUMMUT04 Human methylmalonyl CoA mutase 77 2e-12

[0097] Methylmalonyl CoA mutase requires vitamin B12 (adenosylcobalamin)as an essential cofactor for activity. One of the difficulties inexpressing active methylmalonyl CoA mutase in a heterologous host isthat the host organism may not provide sufficient, if any, amounts ofthis cofactor. Work on the expression of methionine synthase, acobalamin-dependent enzyme, in E. coli, a host that does not synthesizecobalamin, has shown that it is possible to express an activecobalamin-dependent enzyme by increasing the rate of cobalamintransport. See Amaratunga et al., 1996, Biochemistry 35: 2453-2463,entitled “A synthetic module for the metH gene permits facilemutagenesis of the cobalamin-binding region of Escherichia colimethionine synthase: initial characterization of seven mutant proteins,”incorporated herein by reference.

[0098] The methods of the present invention include the step ofincreasing the availability of cobalamin for the heterologous expressionof active methylmalonyl CoA mutase in certain hosts, e.g. E. coli. Inparticular, these methods incorporate growing cells in a media thatcontains hydroxocobalamin and/or other nutrients, as described inAmaratunga et al., supra. Additional methods for increasing theavailability of cobalamin include constitutive and/or over-expression ofvitamin B12 transporter proteins and/or their regulators.

[0099] A suitable methylmalonyl CoA epimerase (5.1.99.1) gene forpurposes of the present invention can be isolated from Streptomycescoelicolor as reported in GenBank locus SC5F2A as gene SC5F2A.13(referred to here as EP5) or from S, coelicolor as reported in GenBanklocus SC6A5 as gene SC6A5.34 (referred to here as EP6). See Redenbach etal., 1996, Mol. Microbiol. 21 (1), 77-96, entitled “A set of orderedcosmids and a detailed genetic and physical map for the 8 MbStreptomyces coelicolor A3(2) chromosome,” incorporated herein byreference. To date, no biochemical characterization of the proteinsencoded by the genes EP5 and EP6 has been carried out; thus, the presentinvention provides a method for using these genes to providemethylmalonyl CoA epimerase activity to a host. That these genes encodeproteins with methylmalonyl CoA epimerase activity is supported by theirhomology to the sequence of a 2-arylpropionyl CoA epimerase from rat.See Reichel et al., 1997, Mol. Pharmacol. 51: 576-582, entitled“Molecular cloning and expression of a 2-arylpropionyl-coenzyme Aepimerase: a key enzyme in the inversion metabolism of ibuprofen,” andShieh & Chen, 1993, J. Biol. Chem. 268: 3487-3493, entitled“Purification and characterization of novel ‘2-arylpropionyl CoAepimerases’ from rat liver cytosol and mitochondria.” Both rat2-arylpropionyl CoA epimerase and methylmalonyl CoA epimerase catalyzethe same stereoisomeric inversion, but with different chemical groupsattached.

[0100] Biochemical characterization of a methylmalonyl CoA epimeraseenzyme purified from Propionibacterium shermanii has been completed. SeeLeadlay, 1981, Biochem. J. 197: 413-419, entitled “Purification andcharacterization of methylmalonyl CoA epimerase from Propionibacteriumshermanii,” Leadlay & Fuller, 1983, Biochem. J. 213: 635-642, entitled“Proton transfer in methylmalonyl CoA epimerase from Propionibacteriumshermanii: Studies with specifically tritiated (2R)-methylmalonyl CoA assubstrate; Fuller & Leadlay, 1983, Biochem. J. 213: 643-650, entitled“Proton transfer in methylmalonyl CoA epimerase from Propionibacteriumshermanii: The reaction of (2R)-methylmalonyl CoA in tritiated water.”The DNA sequence of the gene coding for this enzyme fromPropionibacterium shermanii is provided by the present invention inisolated and recombinant form and is incorporated into expressionvectors and host cells of the invention. Suitable methylmalonyl CoAepimerase genes can be isolated from a BLAST search using the P.shermanii sequence provided in Example 1, below. Preferred epimerases inaddition to the P. shermanii epimerase include gene identified byhomology with the P. shermanii sequence located on cosmid 8F4 from theS. coelicolor genome sequencing project and the B. subtilis epimerasedescribed by Haller et al., 2000, Biochemistry 39 (16): 4622-4629,incorporated herein by reference.

[0101] One can also make Smethylmalonyl CoA from R-methylmalonyl CoAutilizing an activity of malonyl CoA decarboxylase A, which convertsR-methylmalonyl CoA to propionyl CoA. As described above, propionyl CoAcan then be converted to Smethylmalonyl CoA by propionyl CoAcarboxylase. A suitable malonyl CoA decarboxylase (4.1.1.9) gene forpurposes of the present invention can be isolated from Saccharopolysporaerythraea as reported in Hsieh & Kolattukudy, 1994, J. Bacteriol. 176:714-724, entitled “Inhibition of erythromycin synthesis by disruption ofmalonyl-coenzyme A decarboxylase gene eryM in Saccharopolysporaerythraea.” Alternatively, suitable malonyl CoA decarboxylase genes canbe isolated from any of the sources noted in the following table ofBLAST search reports or by additional BLAST searches.

Results of BLAST Search of NCBI Database for Malonyl CoA DecarboxylaseMalonyl CoA Decarboxylase (DC)

[0102] gb|L05192|SERMALCOAD S. erythraea malonyl 664 0.0 (querysequence)

[0103] emb|AL022268|SC4H2 Streptomyces coelicolor cosmid 4H2 128 3e-28

[0104] emb|Z75555|MTCY02B10 Mycobacterium tuberculosis H37Rv 109 1e-22

[0105] gb|AD000018|MSGY151 Mycobacterium tuberculosis sequence 109 1e-22

[0106] gb|AF141323.1|AF141323 Shigella flexneri SHI-2 95 5e-18

[0107] emb|X76100|ECIUC E.coli plasmid iucA, iucB and iucC genes 923e-17

[0108] emb|AL116808.1|CNS01DGW Botrytis cinerea strain T4 cDNA 88 5e-16

[0109] gb|AF110737.1|AF110737 Sinorhizobium meliloti strain 2011 849e-15

[0110] emb|AL109846.1|SPBC17G9 S.pombe chromosome II cosmid c17G9 717e-11

[0111] gb|L06163|PSEAAC Pseudomonas fluorescens aminoglycoside 70 1e-10

[0112] A suitable propionyl CoA carboxylase (6.4.1.3) gene for purposesof the present invention can be isolated from Streptomyces coelicolor asreported in GenBank locus AF113605 (pccB), AF113604 (accA2) and AF113603(accA1) by H. C. Gramajo and colleagues. The propionyl CoA carboxylasegene product requires biotin for activity. If the host cell does notmake biotin, then the genes for biotin tranport can be transferred tothe host cell. Even if the host cell makes or transports biotin, theendogenous biotin transferase enzyme may not have sufficient activity(whether due to specificity constraints or other reasons) to biotinylatethe propionyl CoA carboxylase at the rate required for high levelprecursor synthesis. In this event, one can simply provide the host cellwith a sufficiently active biotin transferase enzyme gene, or if thereis an endongenous transferase gene, such as the birA gene in E. coli,one can simply overexpress that gene by recombinant methods. Manyadditional genes coding for propionyl CoA carboxylases, or acetyl CoAcarboxylases with relaxed substrate specificity that includespropionate, have been reported and can be used as sources for this gene,as shown in the following table.

Results of BLAST Search of NCBI Database for Propionyl CoA CarboxylasePropionyl CoA Carboxylase (pccB)

[0113] gb|AF113605.1|AF113605 S. coelicolor propionyl 1035 0.0 (querysequence)

[0114] emb|X92557|SEPCCBBCP S.erythraea pccB, bcpA2, and orfX 800 0.0

[0115] emb|Z92771|MTCY71 Mycobacterium tuberculosis H37Rv 691 0.0

[0116] dbj|AB018531|AB018531 Corynebacterium glutamicum dtsR1 686 0.0

[0117] gb|U00012|U00012 Mycobacterium leprae cosmid B1308 686 0.0

[0118] dbj|AB018530|AB018530 Corynebacterium glutamicum dtsR gene 612e-174

[0119] gb|AE001742.1|AE001742 Thermotoga maritima section 54 610 e-173

[0120] emb|AJ002015|PMAJ2015 Propionigenium modestum mmdD 589 e-167

[0121] dbj|AB007000|AB007000 Myxococcus xanthus MxppcB gene 588 e-166

[0122] gb|L48340|MTBKATA Methylobacterium extorquens catalase 588 e-166

[0123] gb|AE0009521|AE000952 Archaeoglobus fulgidus section 155 572e-162

[0124] dbj|AP000005|AP000005 Pyrococcus horikoshii OT3 genomic 570 e-161

[0125] emb|AJ248285.1|CNSPAX03 Pyrococcus abyssi complete genome 570e-161

[0126] emb|AL031124|SC1C2 Streptomyces coelicolor cosmid 1C2 563 e-159

[0127] gb|L222081|VEIMCDC Veillonella parvula methylmalonyl CoA 558e-157

[0128] gb|AF080235|AF080235 Streptomyces cyanogenus landomycin 552 e-155

[0129] emb|AJ235272|RPXX03 Rickettsia prowazekii strain Madrid E 545e-153

[0130] dbj AB000886|AB000886 Sus scrofa mRNA for Propionyl CoA 539 e-152

[0131] ref|NM_(—)000532.1|PCCB| Homo sapiens propionyl Coenzyme A 538e-151

[0132] emb|X73424|HSPCCBA Homo sapiens gene for propionyl CoA 538 e-151

[0133] gb|M14634|RATPCCB Rat mitochondrial propionyl CoA 535 e-150

[0134] gb|S67325|S67325 propionyl CoA carboxylase beta subunit 531 e-149

[0135] gb|U56964|CELF52E4 Caenorhabditis elegans cosmid F52E4 367 e-143

[0136] emb|Z99116|BSUB0013 Bacillus subtilis complete genome 494 e-138

[0137] dbj|D844321|BACJH642 Bacillus subtilis DNA, 283 Kb region 494e-138

[0138] gb|AF042099|AF042099 Sulfolobus metallicus putative 486 e-136

[0139] emb|AL022076.1|MTV026 Mycobacterium tuberculosis H37Rv 483 e-135

[0140] gb|L04196|PRSTRANSC Propionibacterium shermanii 383 e-104

[0141] emb|AL023635.1|MLCB1243 Mycobacterium leprae cosmid B1243 3561e-96

[0142] emb|Z70692.1|MTCY427 Mycobacterium tuberculosis H37Rv 353 1e-95

[0143] gb|L78825|MSGB1723CS Mycobacterium leprae cosmid B1723 DNA 3194e-93

[0144] gb|M95713|RERCOABETA Rhodococcus erythropolis 340 5e-92

[0145] emb|Z99113|BSUB0010 Bacillus subtilis complete genome 325 2e-87

[0146] gb|U94697|CCU94697 Caulobacter crescentus DNA topoisomerase 2706e-71

[0147] emb|Z95556|MTCY07A7 Mycobacterium tuberculosis H37Rv 253 9e-66

[0148] emb|Y07660|MTACCBC M.tuberculosis accBC gene 231 6e-59

[0149] emb|Z79700|MTCY10D7 Mycobacterium tuberculosis H37Rv 229 2e-58

[0150] dbj|AB018557.1|AB018557 Streptomyces griseus cyaA gene 228 5e-58

[0151] gb|U46844|MSU46844 Mycobacterium smegmatis catalase 209 2e-52

[0152] emb|Z19555.1|CEF02A9 Caenorhabditis elegans cosmid F02A9 1059e-51

[0153] gb|M13573|HUMPCCB Human propionyl CoA carboxylase beta 194 5e-48

[0154] gb|AF030576|AF030576 Acidaminococcus fermentans 170 9e-41

[0155] emb|Y13917|BSY13917 Bacillus subtilis ppsE, yngL, yngK 149 2e-34

[0156] emb|X69435|AFGCDA A.fermentans GCDA gene for 107 1e-21

[0157] emb|Z82368|RPZ82368 R.prowazekii genomic DNA fragment 93 2e-17

[0158] gb|AF025469|CELW09B6 Caenorhabditis elegans cosmid W09B6 78 5e-13

[0159] gb|U87980|MRU87980 Malonomonas rubra putative IS-element 78 7e-13

[0160] gb|AE001518|AE001518 Helicobacter pylori, strain J99 75 6e-12

[0161] gb|AE000604.1|AE000604 Helicobacter pylori 26695 section 82 758e-12

[0162] gb|U89347|ACU89347 Acinetobacter calcoaceticus malonate 74 1e-11

[0163] emb|AL021961|ATF28A23 Arabidopsis thaliana DNA 61 2e-11

[0164] gb|AE001591|AE001591 Chlamydia pneumoniae section 7 73 2e-11

[0165] emb|Z46886|UMACCGEN U.maydis ACC gene for acetyl coa 71 1e-10

[0166] gb|U86128|SSPCCB1 Sus scrofa propionyl CoA carboxylase B 70 2e-10

[0167] emb|AJ006497|HSA006497 Homo sapiens PCCB gene, exons 11 70 2e-10

[0168] gb|AE001301|AE001301 Chlamydia trachomatis section 28 69 5e-10

[0169] gb|U32724|U32724 Haemophilus influenzae Rd section 39 68 8e-10

[0170] gb|U04358|PSU04358 Pseudomonas syringae pv. syringae Y30 68 8e-10

Propionyl CoA Carboxylase (accA2)

[0171] gb|AF113604.1|AF113604 S. coelicolor putative 1101 0.0 (querysequence)

[0172] gb|AF113603.1|AF113603 Streptomyces coelicolor putative 1090 0.0

[0173] gb|AF126429.1|AF126429 Streptomyces venezuelae JadJ 967 0.0

[0174] emb|Z92771|MTCY71 Mycobacterium tuberculosis H37Rv 758 0.0

[0175] emb|X92557|SEPCCBBCP S.erythraea pccB, bcpA2, and orfX genes 7530.0

[0176] emb|X92556|SEHGTABCP S.erythraea hgtA, bcpA1, and orf122 753 0.0

[0177] gb|U00012|U00012 Mycobacterium leprae cosmid B1308 746 0.0

[0178] emb|X63470|MLBCCPG M.leprae gene for biotin carboxyl 743 0.0

[0179] gb|U35023|CGU35023 Corynebacterium glutamicum thiosulfate 695 0.0

[0180] gb|U24659|SVU24659 Streptomyces venezuelae glucose 599 e-170

[0181] gb|AE000742|AE000742 Aquifex aeolicus section 74 413 e-113

[0182] gb|U67563|U67563 Methanococcus jannaschii section 105 405 e-111

[0183] gb|L36530|MQSPYRCARB Aedes aegypti pyruvate carboxylase 400 e-110

[0184] gb|AF132152.1|AF132152 Drosophila melanogaster clone 396 e-108

[0185] gb|L09192|MUSMPYR Mus musculus pyruvate carboxylase 393 e-107

[0186] gb|U36585|RNU36585 Rattus norvegicus pyruvate carboxylase 391e-107

[0187] gb|U32314|RNU32314 Rattus norvegicus pyruvate carboxylase 391e-107

[0188] gb|L14862|ANAACCC Anabaena sp. (PCC 7120) 49.1 kDa biotin 388e-106

[0189] gb|U59234|SPU59234 Synechococcus PCC7942 biotin 387 e-106

[0190] gb|U04641|HSU04641 Human pyruvate carboxylase (PC) mRNA 387 e-106

[0191] ref|NM_(—)000920.1|PC| Homo sapiens pyruvate carboxylase (PC) 386e-105

[0192] gb|AE001090|AE001090 Archaeoglobus fulgidus section 17 383 e-104

[0193] dbj|D84432|BACJH642 Bacillus subtilis DNA, 283 Kb region 382e-104

[0194] emb|Z99116|BSUB0013 Bacillus subtilis complete genome 382 e-104

[0195] gb|AE000942|AE000942 Methanobacterium thermoautotrophicum 382e-104

[0196] gb|S72370|S72370 pyruvate carboxylase human, kidney 380 e-104

[0197] dbj|D64001|SYCCPNC Synechocystis sp. PCC6803 complete 379 e-103

[0198] gb|L14612|PSEACCBC Pseudomonas aeruginosa biotin carboxyl 376e-103

[0199] gb|U32778|U32778 Haemophilus influenzae Rd section 93 375 e-102

[0200] emb|Z36087|SCYBR218C S.cerevisiae chromosome 11 374 e-102

[0201] gb|U35647|SCU35647 Saccharomyces cerevisiae pyruvate 374 e-102

[0202] gb|J03889|YSCPCB Yeast (S.cerevisiae) pyruvate carboxylase 374e-102

[0203] gb|U90879|ATU90879 Arabidopsis thaliana biotin carboxylase 374e-102

[0204] emb|Z72584|SCYGL062W S.cerevisiae chromosome VII 374 e-102

[0205] emb|X59890|SCPYC2G S.cerivisiae PYC2 gene for pyruvate 373 e-102

[0206] gb|AE000749|AE000749 Aquifex aeolicus section 81 371 e-101

[0207] gb|AE001286|AE001286 Chlamydia trachomatis section 13 370 e-101

[0208] gb|AE001604|AE001604 Chlamydia pneumoniae section 20 369 e-100

[0209] gb|AF007100|AF007100 Glycine max biotin carboxylase 368 e-100

[0210] emb|Z95556|MTCY07A7 Mycobacterium tuberculosis H37Rv 367 e-100

[0211] emb|Z19549|MTBCARBCP M.tuberculosis gene for biotin 367 e-100

[0212] gb|AF068249|AF068249 Glycine max biotin carboxylase 366 1e-99

[0213] gb|L38260|TOBBCSO Nicotiana tabacum acetyl CoA 363 7e-99

[0214] gb|U36245|BSU36245 Bacillus subtilis biotin carboxyl 362 2e-98

[0215] gb|AF097728|AF097728 Aspergillus terreus pyruvate 361 3e-98

[0216] emb|AJ235272|RPXX03 Rickettsia prowazekii strain Madrid E 3601e-97

[0217] dbj|D83706|D83706 Bacillus stearothermophilus DNA 360 1e-97

[0218] gb|AE000744|AE000744 Aquifex aeolicus section 76 358 3e-97

[0219] emb|AL109846.1|SPBC17G9 S.pombe chromosome II 356 1e-96

[0220] dbj|D78170|D78170 Yeast DNA for pyruvate carboxylase 353 1e-95

[0221] gb|M79446|ECOFABG Escherichia coli biotin carboxylase gene 3522e-95

[0222] gb|M83198|ECOFABEGF Escherichia coli biotin carboxyl 352 2e-95

[0223] gb|AE000404|AE000404 Escherichia coli K-12 MG1655 352 2e-95

[0224] gb|U18997.1|ECOUW67 Escherichia coli K-12 chromosomal 352 2e-95

[0225] gb M80458|ECOACOAC E.coli biotin carboxylase and biotin 352 2e-95

[0226] gb U51439|REU51439 Rhizobium etli pyruvate carboxylase 351 5e-95

[0227] emb|Y13917|BSY13917 Bacillus subtilis ppsE, yngL, yngK 348 3e-94

[0228] emb|Z99113|BSUB0010 Bacillus subtilis complete genome 348 3e-94

[0229] gb|AE001274.1|AE001274 Leishmania major chromosome 1 347 6e-94

[0230] gb AF042099|AF042099 Sulfolobus metallicus putative 346 1e-93

[0231] emb|Z81052.1|CED2023 Caenorhabditis elegans cosmid D2023 1623e-92

[0232] emb|Z79700|MTCY10D7 Mycobacterium tuberculosis H37Rv 341 4e-92

[0233] emb|Z99111|BSUB0008 Bacillus subtilis complete genome 340 1e-91

[0234] gb|U12536|ATU12536 Arabidopsis thaliana 3-methylcrotonyl 3384e-91

[0235] emb|Y11106|PPPYC1 P.pastoris PYC1 gene 338 4e-91

[0236] gb|AE001529|AE001529 Helicobacter pylori, strain J99 334 5e-90

[0237] gb|AE000553.1|AE000553 Helicobacter pylori 26695333 7e-90

[0238] emb|Y09548|CGPYC Corynebacterium glutamicum pyc gene 333 1e-89

[0239] gb|AF038548|AF038548 Corynebacterium glutamicum pyruvate 3331e-89

[0240] ref|NM_(—)000282.1|PCCA| Homo sapiens Propionyl Coenzyme 3331e-89

[0241] gb|M22631|RATPCOA Rat alpha-propionyl CoA carboxylase 332 2e-89

[0242] gb|U08469 GMU08469 Glycine max 3-methylcrotonyl CoA 328 3e-88

[0243] emb Z83018 MTCY349 Mycobacterium tuberculosis H37Rv 318 4e-85

[0244] emb|AJ243652.1|PFL243652 Pseudomonas fluorescens uahA gene 3161e-84

[0245] emb|Z36077|SCYBR208C S.cerevisiae chromosome II 312 2e-83

[0246] gb|M64926|YSCUAMD Yeast urea amidolyase (DUR1.2) gene 311 5e-83

[0247] emb|Z97025|BSZ97025 Bacillus subtilis nprE, yla[A,B,C,D,E,F 3001e-79

[0248] emb|Z81074.1|CEF32B6 Caenorhabditis elegans cosmid F32B6 1317e-78

[0249] gb|U00024|MTU00024 Mycobacterium tuberculosis cosmid tbc2 2847e-75

[0250] gb AD000009|MSGY2 Mycobacterium tuberculosis sequence 284 7e-75

[0251] gb U34393|GMU34393 Glycine max acetyl CoA carboxylase 259 2e-67

[0252] gb|U49829|CELF27D9 Caenorhabditis elegans cosmid F27D9 186 4e-59

[0253] emb|AJ010111.1|BCE010111 Bacillus cereus pycA, ctaA, ctaB 2085e-52

[0254] gb|U19183|ZMU19183 Zea mays acetyl-coenzyme A carboxylase 2085e-52

[0255] gb|U10187|TAU10187 Triticum aestivum Tam 107 206 2e-51

[0256] gb|AF029895|AF029895 Triticum aestivum acetyl-coenzyme A 2055e-51

[0257] gb|J03808|RATACACA Rat acetyl-coenzyme A carboxylase mRNA 2048e-51

[0258] emb|X80045|OAACOAC O.aries mRNA for acetyl CoA carboxylase 2031e-50

[0259] emb|X68968|HSACOAC H.sapiens mRNA for acetyl CoA 203 2e-50

[0260] emb|AJ132890.1|BTA132890 Bos taurus mRNA for acetyl 202 2e-50

[0261] gb|J03541|CHKCOACA Chicken acetyl CoA carboxylase mRNA 202 3e-50

[0262] dbj|D34630|ATHACCRNA Arabidopsis thaliana mRNA 199 2e-49

[0263] gb|L25042|ALFACCASE Medicago sativa acetyl CoA carboxylasel985e-49

[0264] emb|Z71631|SCYNR016C S.cerevisiae chromosome XIV 193 2e-47

[0265] gb|M92156|YSCFAS3A Saccharomyces cerevisiae acetyl CoA 193 2e-47

[0266] emb|Z49809|SC8261X S.cerevisiae chromosome XIII cosmid 8261 1923e-47

[0267] emb|Z22558|SCHFA1GN S.cerevisiae HFA1 gene 192 3e-47

[0268] dbj|D78165|D78165 Saccharomyces cerevisiae DNA 192 3e-47

[0269] emb|Z46886|UMACCGEN U.maydis ACC gene for acetyl coa 190 1e-46

[0270] ref|NM_(—)001093.1|ACACB| Homo sapiens acetyl Coenzyme A 1815e-44

Propionyl CoA Carboxylase (accA1)

[0271] gb|AF113603.1|AF113603 S. coelicolor putative 1101 0.0 (querysequence)

[0272] gb AF113604.1|AF113604 Streptomyces coelicolor putative 1090 0.0

[0273] gb AF126429.1|AF126429 Streptomyces venezuelae JadJ (jadJ) 9670.0

[0274] emb Z92771|MTCY71 Mycobacterium tuberculosis H37Rv 758 0.0

[0275] emb|X92557|SEPCCBBCP S.erythraea pccB, bcpA2, and orfX genes 7530.0

[0276] emb|X92556|SEHGTABCP S.erythraea hgtA, bcpA1, and orf122 753 0.0

[0277] gb|U00012|U00012 Mycobacterium leprae cosmid B1308 745 0.0

[0278] emb|X63470|MLBCCPG M.leprae gene for biotin carboxyl 742 0.0

[0279] gb|U35023|CGU35023 Corynebacterium glutamicum thiosulfate 694 0.0

[0280] gb|U2465 |SVU24659 Streptomyces venezuelae glucose 596 e-169

[0281] gb AE000742|AE000742 Aquifex aeolicus section 74 417 e-115

[0282] gb|U67563|U67563 Methanococcus jannaschii section 105 413 e-114

[0283] gb|L36530|MQSPYRCARB Aedes aegypti pyruvate carboxylase 404 e-111

[0284] gb AF132152.1|AF132152 Drosophila melanogaster clone 400 e-110

[0285] gb|L09192|MUSMPYR Mus musculus pyruvate carboxylase 397 e-109

[0286] gb U36585|RNU36585 Rattus norvegicus pyruvate carboxylase 395e-108

[0287] gb|U32314|RNU32314 Rattus norvegicus pyruvate carboxylase 395e-108

[0288] gb|L14862|ANAACCC Anabaena sp. (PCC 7120) 49.1 kDa biotin 394e-108

[0289] gb|U04641|HSU04641 Human pyruvate carboxylase (PC) mRNA 391 e-107

[0290] gb|U59234|SPU59234 Synechococcus PCC7942 biotin carboxylase 391e-107

[0291] ref|NM_(—)000920.1|PC| Homo sapiens pyruvate carboxylase (PC) 390e-107

[0292] gb AE001090|AE001090 Archaeoglobus fulgidus section 17 389 e-106

[0293] gb|AE000942|AE000942 Methanobacterium thermoautotrophicum 386e-105

[0294] gb|S72370|S72370 pyruvate carboxylase human, kidney 384 e-105

[0295] dbj|D84432|BACJH642 Bacillus subtilis DNA, 283 Kb region 383e-105

[0296] emb|Z99116|BSUB0013 Bacillus subtilis complete genome 383 e-105

[0297] dbj|D64001|SYCCPNC Synechocystis sp. PCC6803 383 e-104

[0298] gb|U35647|SCU35647 Saccharomyces cerevisiae pyruvate 382 e-104

[0299] emb|Z36087|SCYBR218C S.cerevisiae chromosome II 382 e-104

[0300] emb Z72584|SCYGL062W S.cerevisiae chromosome VII 381 e-104

[0301] gb|J03889|YSCPCB Yeast (S.cerevisiae) pyruvate carboxylase 381e-104

[0302] gb|L14612|PSEACCBC Pseudomonas aeruginosa biotin carboxyl 381e-104

[0303] emb|X59890|SCPYC2G S.cerivisiae PYC2 gene for pyruvate 381 e-104

[0304] gb|U32778|U32778 Haemophilus influenzae Rd section 93 380 e-104

[0305] gb|U90879|ATU90879 Arabidopsis thaliana biotin carboxylase 377e-103

[0306] gb|AE000749|AE000749 Aquifex aeolicus section 81 of 109 377 e-103

[0307] gb|AE001286|AE001286 Chlamydia trachomatis section 13 375 e-102

[0308] gb|AE001604|AE001604 Chlamydia pneumoniae section 20 374 e-102

[0309] gb|AF007100|AF007100 Glycine max biotin carboxylase 372 e-101

[0310] emb|Z95556|MTCY07A7 Mycobacterium tuberculosis H37Rv 369 e-100

[0311] emb|Z19549|MTBCARBCP M.tuberculosis gene for biotin 369 e-100

[0312] gb|AF068249|AF068249 Glycine max biotin carboxylase 369 e-100

[0313] gb|L38260|TOBBCSO Nicotiana tabacum acetyl CoA 367 e-100

[0314] gb|AF097728|AF097728 Aspergillus terreus pyruvate 366 1e-99

[0315] gb|AE000744|AE000744 Aquifex aeolicus section 76 of 109 364 4e-99

[0316] dbj|D83706|D83706 Bacillus stearothermophilus DNA 363 7e-99

[0317] gb|U36245|BSU36245 Bacillus subtilis biotin carboxyl 363 7e-99

[0318] emb|AL109846.1|SPBC17G9 S.pombe chromosome II 362 2e-98

[0319] emb|AJ235272|RPXX03 Rickettsia prowazekii strain Madrid E 3613e-98

[0320] dbj|D78170|D78170 Yeast DNA for pyruvate carboxylase 359 2e-97

[0321] gb|M80458|ECOACOAC E.coli biotin carboxylase and biotin 358 3e-97

[0322] gb|M79446|ECOFABG Escherichia coli biotin carboxylase gene 3583e-97

[0323] gb|M83198|ECOFABEGF Escherichia coli biotin carboxyl 358 3e-97

[0324] gb|AE000404|AE000404 Escherichia coli K-12 MG1655 358 3e-97

[0325] gb|U18997.1|ECOUW67 Escherichia coli K-12 chromosomal 358 3e-97

[0326] gb|U51439|REU51439 Rhizobium etli pyruvate carboxylase 355 3e-96

[0327] emb|Y13917|BSY13917 Bacillus subtilis ppsE, yngL, yngK, 354 4e-96

[0328] emb|Z99113|BSUB0010 Bacillus subtilis complete genome 354 4e-96

[0329] gb|AE001274.1|AE001274 Leishmania major chromosome 1 351 3e-95

[0330] gb|AF042099|AF042099 Sulfolobus metallicus putative 350 9e-95

[0331] emb|Z79700|MTCY10D7 Mycobacterium tuberculosis H37Rv 347 6e-94

[0332] emb|Z81052.1|CED2023 Caenorhabditis elegans cosmid D2023 1681e-93

[0333] emb|Y11106|PPPYC1 P.pastoris PYC1 gene 345 2e-93

[0334] emb|Z99111|BSUB0008 Bacillus subtilis complete genome 343 8e-93

[0335] ref|NM_(—)000282.1|PCCA| Homo sapiens Propionyl Coenzyme 3406e-92

[0336] gb|M22631|RATPCOA Rat alpha-propionyl CoA carboxylase 340 1e-91

[0337] gb|U12536|ATU12536 Arabidopsis thaliana 3-methylcrotonyl 3392e-91

[0338] emb|Y09548|CGPYC Corynebacterium glutamicum pyc gene 338 4e-91

[0339] gb|AF038548|AF038548 Corynebacterium glutamicum pyruvate 3384e-91

[0340] gb|AE001529|AE001529 Helicobacter pylori, strain J99 337 8e-91

[0341] gb|AE000553.1|AE000553 Helicobacter pylori 26695 336 1e-90

[0342] gb|U08469|GMU08469 Glycine max 3-methylcrotonyl CoA 329 2e-88

[0343] emb|AJ243652.1|PFL243652 Pseudomonas fluorescens uahA gene 3231e-86

[0344] emb|Z83018|MTCY349 Mycobacterium tuberculosis H37Rv 321 3e-86

[0345] emb|Z36077|SCYBR208C S.cerevisiae chromosome II 314 5e-84

[0346] gb|M64926|YSCUAMD Yeast urea amidolyase (DUR1.2) gene 312 2e-83

[0347] emb|Z97025|BSZ97025 Bacillus subtilis nprE, yla[A, B, C, D, E,303 1e-80

[0348] emb|Z81074.1|CEF32B6 Caenorhabditis elegans cosmid F32B6 1301e-78

[0349] gb|U00024|MTU00024 Mycobacterium tuberculosis cosmid tbc2 2876e-76

[0350] gb|AD000009|MSGY2 Mycobacterium tuberculosis sequence 287 6e-76

[0351] gb|U34393|GMU34393 Glycine max acetyl CoA carboxylase 262 3e-68

[0352] gb|U49829|CELF27D9 Caenorhabditis elegans cosmid F27D9 190 2e-61

[0353] gb|U10187|TAU10187 Triticum aestivum Tam 107 213 2e-53

[0354] gb|U19183|ZMU19183 Zea mays acetyl-coenzyme A carboxylase 2123e-53

[0355] emb|AJ010111.1|BCE010111 Bacillus cereus pycA, ctaA, ctaB 2124e-53

[0356] gb|AF029895|AF029895 Triticum aestivum acetyl-coenzyme 209 2e-52

[0357] gb|J03808|RATACACA Rat acetyl-coenzyme A carboxylase 205 4e-51

[0358] emb|X80045|OAACOAC O.aries mRNA for acetyl CoA 205 5e-51

[0359] emb|X68968|HSACOAC H.sapiens mRNA for acetyl CoA 204 8e-51

[0360] dbj|D34630|ATHACCRNA Arabidopsis thaliana mRNA 203 1e-50

[0361] emb|AJ132890.1|BTA132890 Bos taurus mRNA for acetyl CoA 203 1e-50

[0362] gb|J03541|CHKCOACA Chicken acetyl CoA carboxylase mRNA 203 1e-50

[0363] gb|L25042|ALFACCASE Medicago sativa acetyl CoA carboxylase 2022e-50

[0364] emb|Z71631|SCYNR016C S.cerevisiae chromosome XIV 196 1e-48

[0365] gb|M92156|YSCFAS3A Saccharomyces cerevisiae acetyl CoA 196 1e-48

[0366] emb|Z49809|SC8261X S.cerevisiae chromosome XIII cosmid 8261 1954e-48

[0367] emb|Z22558|SCHFA1GN S.cerevisiae HFA1 gene 195 4e-48

[0368] dbj|D78165|D78165 Saccharomyces cerevisiae DNA 195 4e-48

[0369] emb|Z46886|UMACCGEN U.maydis ACC gene for acetyl coa 188 5e-46

[0370] gb|L20784|CCXACOAC Cyclotella cryptica acetyl CoA 182 2e-44

[0371] Those of skill in the art will recognize that, due to thedegenerate nature of the genetic code, a variety of DNA compoundsdiffering in their nucleotide sequences can be used to encode a givenamino acid sequence of the invention. The native DNA sequence encodingthe biosynthetic enzymes in the tables above are referenced hereinmerely to illustrate a preferred embodiment of the invention, and theinvention includes DNA compounds of any sequence that encode the aminoacid sequences of the polypeptides and proteins of the enzymes utilizedin the methods of the invention. In similar fashion, a polypeptide cantypically tolerate one or more amino acid substitutions, deletions, andinsertions in its amino acid sequence without loss or significant lossof a desired activity. The present invention includes such polypeptideswith alternate amino acid sequences, and the amino acid sequencesencoded by the DNA sequences shown herein merely illustrate preferredembodiments of the invention.

[0372] Thus, in an especially preferred embodiment, the presentinvention provides DNA molecules in the form of recombinant DNAexpression vectors or plasmids, as described in more detail below, thatencode one or more precursor biosynthetic enzymes. Generally, suchvectors can either replicate in the cytoplasm of the host cell orintegrate into the chromosomal DNA of the host cell. In either case, thevector can be a stable vector (i.e., the vector remains present overmany cell divisions, even if only with selective pressure) or atransient vector (i.e., the vector is gradually lost by host cells withincreasing numbers of cell divisions). The invention provides DNAmolecules in isolated (i.e., not pure, but existing in a preparation inan abundance and/or concentration not found in nature) and purified(i.e., substantially free of contaminating materials or substantiallyfree of materials with which the corresponding DNA would be found innature) form.

[0373] In one important embodiment, the invention provides methods forthe heterologous expression of one or more of the biosynthetic genesinvolved in S-methylmalonyl CoA biosynthesis and recombinant DNAexpression vectors useful in the method. Thus, included within the scopeof the invention are recombinant expression vectors that include suchnucleic acids. The term expression vector refers to a nucleic acid thatcan be introduced into a host cell or cell-free transcription andtranslation system. An expression vector can be maintained permanentlyor transiently in a cell, whether as part of the chromosomal or otherDNA in the cell or in any cellular compartment, such as a replicatingvector in the cytoplasm. An expression vector also comprises a promoterthat drives expression of an RNA, which typically is translated into apolypeptide in the cell or cell extract. For efficient translation ofRNA into protein, the expression vector also typically contains aribosome-binding site sequence positioned upstream of the start codon ofthe coding sequence of the gene to be expressed. Other elements, such asenhancers, secretion signal sequences, transcription terminationsequences, and one or more marker genes by which host cells containingthe vector can be identified and/or selected, may also be present in anexpression vector. Selectable markers, i.e., genes that conferantibiotic resistance or sensitivity, are preferred and confer aselectable phenotype on transformed cells when the cells are grown in anappropriate selective medium.

[0374] The various components of an expression vector can vary widely,depending on the intended use of the vector and the host cell(s) inwhich the vector is intended to replicate or drive expression.Expression vector components suitable for the expression of genes andmaintenance of vectors in E. coli, yeast, Streptomyces, and othercommonly used cells are widely known and commercially available. Forexample, suitable promoters for inclusion in the expression vectors ofthe invention include those that function in eucaryotic or procaryotichost cells. Promoters can comprise regulatory sequences that allow forregulation of expression relative to the growth of the host cell or thatcause the expression of a gene to be turned on or off in response to achemical or physical stimulus. For E. coli and certain other bacterialhost cells, promoters derived from genes for biosynthetic enzymes,antibiotic-resistance conferring enzymes, and phage proteins can be usedand include, for example, the galactose, lactose (lac), maltose,tryptophan (trp), beta-lactamase (bla), bacteriophage lambda PL, and T5promoters. In addition, synthetic promoters, such as the tac promoter(U.S. Pat. No. 4,551,433), can also be used. For E. coli expressionvectors, it is useful to include an E. coli origin of replication, suchas from pUC, p1P, p1I, and pBR.

[0375] Thus, recombinant expression vectors contain at least oneexpression system, which, in turn, is composed of at least a portion ofPKS and/or other biosynthetic gene coding sequences operably linked to apromoter and optionally termination sequences that operate to effectexpression of the coding sequence in compatible host cells. The hostcells are modified by transformation with the recombinant DNA expressionvectors of the invention to contain the expression system sequenceseither as extrachromosomal elements or integrated into the chromosome.The resulting host cells of the invention are useful in methods toproduce PKS enzymes as well as polyketides and antibiotics and otheruseful compounds derived therefrom.

[0376] Preferred host cells for purposes of selecting vector componentsfor expression vectors of the present invention include fungal hostcells such as yeast and procaryotic host cells such as E. coli, butmammalian host cells can also be used. In hosts such as yeasts, plants,or mammalian cells that ordinarily do not produce polyketides, it may benecessary to provide, also typically by recombinant means, suitableholo-ACP synthases to convert the recombinantly produced PKS tofunctionality. Provision of such enzymes is described, for example, inPCT publication Nos. WO 97/13845 and 98/27203, each of which isincorporated herein by reference.

[0377] The recombinant host cells of the invention can express all ofthe polyketide biosynthetic genes or only a subset of the same. Forexample, if only the genes for a PKS are expressed in a host cell thatotherwise does not produce polyketide modifying enzymes (such ashydroxylation, epoxidation, or glycosylation enzymes) that can act onthe polyketide produced, then the host cell produces unmodifiedpolyketides, called macrolide aglycones. Such macrolide aglycones can behydroxylated and glycosylated by adding them to the fermentation of astrain such as, for example, Streptomyces antibioticus orSaccharopolyspora erythraea, that contains the requisite modificationenzymes.

[0378] There are a wide variety of diverse organisms that can modifymacrolide aglycones to provide compounds with, or that can be readilymodified to have, useful activities. For example, Saccharopolysporaerythraea can convert 6-dEB to a variety of useful compounds. Theerythronolide 6-dEB is converted by the eryF gene product toerythronolide B, which is, in turn, glycosylated by the eryB geneproduct to obtain 3-O-mycarosylerythronolide B, which containsL-mycarose at C-3. The enzyme eryC gene product then converts thiscompound to erythromycin D by glycosylation with D-desosamine at C-5.Erythromycin D, therefore, differs from 6-dEB through glycosylation andby the addition of a hydroxyl group at C-6. Erythromycin D can beconverted to erythromycin B in a reaction catalyzed by the eryG geneproduct by methylating the L-mycarose residue at C-3. Erythromcyin D isconverted to erythromycin C by the addition of a hydroxyl group at C-12in a reaction catalyzed by the eryK gene product. Erythromycin A isobtained from erythromycin C by methylation of the mycarose residue in areaction catalyzed by the eryG gene product. The unmodified polyketidesprovided by the present invention, such as, for example, 6-dEB producedin E. coli, can be provided to cultures of S. erythraea and converted tothe corresponding derivatives of erythromycins A, B, C, and D inaccordance with the procedure provided in the examples below. To ensurethat only the desired compound is produced, one can use an S. erythraeaeryA mutant that is unable to produce 6-dEB but can still carry out thedesired conversions (Weber et al., 1985, J. Bacteriol. 164(1): 425-433).Also, one can employ other mutant strains, such as eryB, eryC, eryG,and/or eryK mutants, or mutant strains having mutations in multiplegenes, to accumulate a preferred compound. The conversion can also becarried out in large fermentors for commercial production.

[0379] Moreover, there are other useful organisms that can be employedto hydroxylate and/or glycosylate the compounds of the invention. Asdescribed above, the organisms can be mutants unable to produce thepolyketide normally produced in that organism, the fermentation can becarried out on plates or in large fermentors, and the compounds producedcan be chemically altered after fermentation. Thus, Streptomycesvenezuelae, which produces picromycin, contains enzymes that cantransfer a desosaminyl group to the C-5 hydroxyl and a hydroxyl group tothe C-12 position. In addition, S. venezuelae contains a glucosylationactivity that glucosylates the 2′-hydroxyl group of the desosaminesugar. This latter modification reduces antibiotic activity, but theglucosyl residue is removed by enzymatic action prior to release of thepolyketide from the cell. Another organism, S. narbonensis, contains thesame modification enzymes as S. venezuelae, except the C-12 hydroxylase.Thus, the present invention provides the compounds produced byhydroxylation and glycosylation of the macrolide aglycones of theinvention by action of the enzymes endogenous to S. narbonensis and S.venezuelae.

[0380] Other organisms suitable for making compounds of the inventioninclude Micromonospora megalomicea, Streptomyces antibioticus, S.fradiae, and S. thermotolerans. M. megalomicea glycosylates the C-3hydroxyl with mycarose, the C-5 hydroxyl with desosamine, and the C-6hydroxyl with megosamine, and hydroxylates the C-6 position. S.antibioticus produces oleandomycin and contains enzymes that hydroxylatethe C-6 and C-12 positions, glycosylate the C-3 hydroxyl with oleandroseand the C-5 hydroxyl with desosamine, and form an epoxide at C-8-C-8a.S. fradiae contains enzymes that glycosylate the C-5 hydroxyl withmycaminose and then the 4′-hydroxyl of mycaminose with mycarose, forminga disaccharide. S. thermotolerans contains the same activities as S.fradiae, as well as acylation activities. Thus, the present inventionprovides the compounds produced by hydroxylation and glycosylation ofthe macrolide aglycones of the invention by action of the enzymesendogenous to M. megalomicea, S. antibioticus, S. fradiae, and S.thermotolerans.

[0381] The present invention also provides methods and geneticconstructs for producing the glycosylated and/or hydroxylated compoundsof the invention directly in the host cell of interest. Thus, the genesthat encode polyketide modification enzymes can be included in the hostcells of the invention. Lack of adequate resistance to a polyketide canbe overcome by providing the host cell with an MLS resistance gene (ermEand mgt/lrm, for example), which confer resistance to several14-membered macrolides (see Cundliffe, 1989, Annu. Rev. Microbiol.43:207-33; Jenkins and Cundliffe, 1991, Gene 108:55-62; and Cundliffe,1992, Gene, 115:75-84, each of which is incorporated herein byreference).

[0382] The recombinant host cells of the invention can be used toproduce polyketides (both macrolide aglycones and their modifiedderivatives) that are naturally occurring or produced by recombinant DNAtechnology. In one important embodiment, the recombinant host cells ofthe invention are used to produce hybrid PKS enzymes. For purposes ofthe invention, a hybrid PKS is a recombinant PKS that comprises all orpart of one or more extender modules, loading module, and/orthioesterase/cyclase domain of a first PKS and all or part of one ormore extender modules, loading module, and/or thioesterase/cyclasedomain of a second PKS.

[0383] Those of skill in the art will recognize that all or part ofeither the first or second PKS in a hybrid PKS of the invention need notbe isolated from a naturally occurring source. For example, only a smallportion of an AT domain determines its specificity. See PCT patentapplication No. WO US99/15047, and Lau et al., infra, incorporatedherein by reference. The state of the art in DNA synthesis allows theartisan to construct de novo DNA compounds of size sufficient toconstruct a useful portion of a PKS module or domain. Thus, the desiredderivative coding sequences can be synthesized using standard solidphase synthesis methods such as those described by Jaye et al., 1984, J.Biol. Chem. 259: 6331, and instruments for automated synthesis areavailable commercially from, for example, Applied Biosystems, Inc. Forpurposes of the invention, such synthetic DNA compounds are deemed to bea portion of a PKS.

[0384] A hybrid PKS for purposes of the present invention can result notonly:

[0385] (i) from fusions of heterologous domain (where heterologous meansthe domains in a module are derived from at least two differentnaturally occurring modules) coding sequences to produce a hybrid modulecoding sequence contained in a PKS gene whose product is incorporatedinto a PKS, but also:

[0386] (ii) from fusions of heterologous module (where heterologousmodule means two modules are adjacent to one another that are notadjacent to one another in naturally occurring PKS enzymes) codingsequences to produce a hybrid coding sequence contained in a PKS genewhose product is incorporated into a PKS,

[0387] (iii) from expression of one or more PKS genes from a first PKSgene cluster with one or more PKS genes from a second PKS gene cluster,and

[0388] (iv) from combinations of the foregoing.

[0389] Various hybrid PKSs of the invention illustrating these variousalternatives are described herein.

[0390] Recombinant methods for manipulating modular PKS genes to makehybrid PKS enzymes are described in U.S. Pat. Nos. 5,672,491; 5,843,718;5,830,750; and 5,712,146; and in PCT publication Nos. 98/49315 and97/02358, each of which is incorporated herein by reference. A number ofgenetic engineering strategies have been used with DEBS to demonstratethat the structures of polyketides can be manipulated to produce novelnatural products, primarily analogs of the erythromycins (see the patentpublications referenced supra and Hutchinson, 1998, Curr Opin Microbiol.1:319-329, and Baltz, 1998, Trends Microbiol. 6:76-83, incorporatedherein by reference).

[0391] These techniques include: (i) deletion or insertion of modules tocontrol chain length, (ii) inactivation of reduction/dehydration domainsto bypass beta-carbon processing steps, (iii) substitution of AT domainsto alter starter and extender units, (iv) addition ofreduction/dehydration domains to introduce catalytic activities, and (v)substitution of ketoreductase KR domains to control hydroxylstereochemistry. In addition, engineered blocked mutants of DEBS havebeen used for precursor directed biosynthesis of analogs thatincorporate synthetically derived starter units. For example, more than100 novel polyketides were produced by engineering single andcombinatorial changes in multiple modules of DEBS. Hybrid PKS enzymesbased on DEBS with up to three catalytic domain substitutions wereconstructed by cassette mutagenesis, in which various DEBS domains werereplaced with domains from the rapamycin PKS (see Schweke et al., 1995,Proc. Nat. Acad. Sci. USA 92, 7839-7843, incorporated herein byreference) or one more of the DEBS KR domains was deleted. Functionalsingle domain replacements or deletions were combined to generate DEBSenzymes with double and triple catalytic domain substitutions (seeMcDaniel et al., 1999, Proc. Nat. Acad. Sci. USA 96, 1846-1851,incorporated herein by reference).

[0392] Methods for generating libraries of polyketides have been greatlyimproved by cloning PKS genes as a set of three or more mutuallyselectable plasmids, each carrying a different wild-type or mutant PKSgene, then introducing all possible combinations of the plasmids withwild-type, mutant, and hybrid PKS coding sequences into the same host(see U.S. patent application Serial No. 60/129,731, filed Apr. 16, 1999,and PCT Pub. No. 98/27203, each of which is incorporated herein byreference). This method can also incorporate the use of a KS1° mutant,which by mutational biosynthesis can produce polyketides made fromdiketide starter units (see Jacobsen et al., 1997, Science 277, 367-369,incorporated herein by reference), as well as the use of a truncatedgene that leads to 12-membered macrolides or an elongated gene thatleads to 16-membered ketolides. Moreover, by utlizing in addition one ormore vectors that encode glycosyl biosynthesis and transfer genes, suchas those of the present invention for megosamine, desosamine,oleandrose, cladinose, and/or mycarose (in any combination), a largecollection of glycosylated polyketides can be prepared.

[0393] The following table lists references describing illustrative PKSgenes and corresponding enzymes that can be utilized in the constructionof the recombinant hybrid PKSs and the corresponding DNA compounds thatencode them. Also presented are various references describing tailoringenzymes and corresponding genes that can be employed in accordance withthe methods of the invention.

[0394] Avermectin

[0395] U.S. Pat. No. 5,252,474 to Merck.

[0396] MacNeil et al., 1993, Industrial Microorganisms: Basic andApplied Molecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp.245-156, A Comparison of the Genes Encoding the Polyketide Synthases forAvermectin, Erythromycin, and Nemadectin.

[0397] MacNeil et al., 1992, Gene 115: 119-125, Complex Organization ofthe Streptomyces avermitilis genes encoding the avermectin polyketidesynthase.

[0398] Candicidin (FR008)

[0399] Hu et al., 1994, Mol. Microbiol. 14: 163-172.

[0400] Epothilone

[0401] PCT Pat. Pub. No. WO 00/031247 to Kosan.

[0402] Erythromycin

[0403] PCT Pub. No. 93/13663 to Abbott.

[0404] U.S. Pat. No. 5,824,513 to Abbott.

[0405] Donadio et al., 1991, Science 252:675-9.

[0406] Cortes et al., Nov. 8, 1990, Nature 348:176-8, An unusually largemultifunctional polypeptide in the erythromycin producing polyketidesynthase of Saccharopolyspora erythraea.

[0407] Glycosylation Enzymes

[0408] PCT Pat. App. Pub. No. 97/23630 to Abbott.

[0409] FK-506

[0410] Motamedi et al., 1998, The biosynthetic gene cluster for themacrolactone ring of the immunosuppressant FK506, Eur. J. biochem. 256:528-534.

[0411] Motamedi et al., 1997, Structural organization of amultifunctional polyketide synthase involved in the biosynthesis of themacrolide immunosuppressant FK506, Eur. J. Biochem. 244: 74-80.

[0412] Methyltransferase

[0413] U.S. Pat. No. 5,264,355, issued Nov. 23, 1993, Methylating enzymefrom Streptomyces MA6858. 31-O-desmethyl-FK506 methyltransferase.

[0414] Motamedi et al., 1996, Characterization of methyltransferase andhydroxylase genes involved in the biosynthesis of the immunosuppressantsFK506 and FK520, J. Bacteriol. 178: 5243-5248.

[0415] FK-520

[0416] PCT Pat. Pub. No. WO 00/020601 to Kosan.

[0417] See also Nielsen et al., 1991, Biochem. 30:5789-96 (enzymology ofpipecolate incorporation).

[0418] Lovastatin

[0419] U.S. Pat. No. 5,744,350 to Merck.

[0420] Narbomycin (and Picromycin)

[0421] PCT Pat. Pub. No. WO 99/61599 to Kosan.

[0422] Nemadectin

[0423] MacNeil et al., 1993, supra.

[0424] Niddamycin

[0425] Kakavas et al., 1997, Identification and characterization of theniddamycin polyketide synthase genes from Streptomyces caelestis, J.Bacteriol. 179: 7515-7522.

[0426] Oleandomycin

[0427] Swan et al., 1994, Characterisation of a Streptomycesantibioticus gene encoding a type I polyketide synthase which has anunusual coding sequence, Mol. Gen. Genet. 242: 358-362.

[0428] PCT Pat. Pub. No. WO 00/026349 to Kosan.

[0429] Olano et al., 1998, Analysis of a Streptomyces antibioticuschromosomal region involved in oleandomycin biosynthesis, which encodestwo glycosyltransferases responsible for glycosylation of themacrolactone ring, Mol. Gen. Genet. 259(3): 299-308.

[0430] Platenolide

[0431] EP Pat. App. Pub. No. 791,656 to Lilly.

[0432] Rapamycin

[0433] Schwecke et al., August 1995, The biosynthetic gene cluster forthe polyketide rapamycin, Proc. Natl. Acad. Sci. USA 92:7839-7843.

[0434] Aparicio et al., 1996, Organization of the biosynthetic genecluster for rapamycin in Streptomyces hygroscopicus: analysis of theenzymatic domains in the modular polyketide synthase, Gene 169: 9-16.

[0435] Rifamycin

[0436] August et al., Feb. 13, 1998, Biosynthesis of the ansamycinantibiotic rifamycin: deductions from the molecular analysis of the rifbiosynthetic gene cluster of Amycolatopsis mediterranei S669, Chemistry& Biology, 5(2): 69-79.

[0437] Soraphen

[0438] U.S. Pat. No. 5,716,849 to Novartis.

[0439] Schupp et al., 1995, J. Bacteriology 177: 3673-3679. A Sorangiumcellulosum (Myxobacterium) Gene Cluster for the Biosynthesis of theMacrolide Antibiotic Soraphen A: Cloning, Characterization, and Homologyto Polyketide Synthase Genes from Actinomycetes.

[0440] Spiramycin

[0441] U.S. Pat. No. 5,098,837 to Lilly.

[0442] Activator Gene

[0443] U.S. Pat. No. 5,514,544 to Lilly.

[0444] Tylosin

[0445] EP Pub. No. 791,655 to Lilly.

[0446] Kuhstoss et al., 1996, Gene 183:231-6., Production of a novelpolyketide through the construction of a hybrid polyketide synthase.

[0447] U.S. Pat. No. 5,876,991 to Lilly.

[0448] Tailoring enzymes

[0449] Merson-Davies and Cundliffe, 1994, Mol. Microbiol. 13: 349-355.Analysis of five tylosin biosynthetic genes from the tylBA region of theStreptomyces fradiae genome.

[0450] As the above Table illustrates, there are a wide variety of PKSgenes that serve as readily available sources of DNA and sequenceinformation for use in constructing the hybrid PKS-encoding DNAcompounds of the invention.

[0451] In constructing hybrid PKSs, certain general methods may behelpful. For example, it is often beneficial to retain the framework ofthe module to be altered to make the hybrid PKS. Thus, if one desires toadd DH and ER functionalities to a module, it is often preferred toreplace the KR domain of the original module with a KR, DH, and ERdomain-containing segment from another module, instead of merelyinserting DH and ER domains. One can alter the stereochemicalspecificity of a module by replacement of the KS domain with a KS domainfrom a module that specifies a different stereochemistry. See Lau etal., 1999, “Dissecting the role of acyltransferase domains of modularpolyketide synthases in the choice and stereochemical fate of extenderunits” Biochemistry 38(5):1643-1651, incorporated herein by reference.One can alter the specificity of an AT domain by changing only a smallsegment of the domain. See Lau et al., supra. One can also takeadvantage of known linker regions in PKS proteins to link modules fromtwo different PKSs to create a hybrid PKS. See Gokhale et al., Apr. 16,1999, Dissecting and Exploiting Intermodular Communication in PolyketideSynthases”, Science 284: 482-485, incorporated herein by reference.

[0452] The hybrid PKS-encoding DNA compounds can be and often arehybrids of more than two PKS genes. Even where only two genes are used,there are often two or more modules in the hybrid gene in which all orpart of the module is derived from a second (or third) PKS gene.

[0453] The invention also provides libraries of PKS genes, PKS proteins,and ultimately, of polyketides, that are constructed by generatingmodifications in a PKS so that the protein complexes produced havealtered activities in one or more respects and thus produce polyketidesother than the natural product of the PKS. Novel polyketides may thus beprepared, or polyketides in general prepared more readily, using thismethod. By providing a large number of different genes or gene clustersderived from a naturally occurring PKS gene cluster, each of which hasbeen modified in a different way from the native cluster, an effectivelycombinatorial library of polyketides can be produced as a result of themultiple variations in these activities. As will be further describedbelow, the metes and bounds of this embodiment of the invention can bedescribed on the polyketide, protein, and the encoding nucleotidesequence levels.

[0454] There are at least five degrees of freedom for constructing ahybrid PKS in terms of the polyketide that will be produced. First, thepolyketide chain length is determined by the number of extender modulesin the PKS, and the present invention includes hybrid PKSs that contain6, as wells as fewer or more than 6, extender modules. Second, thenature of the carbon skeleton of the PKS is determined by thespecificities of the acyl transferases that determine the nature of theextender units at each position, e.g., malonyl, methylmalonyl,ethylmalonyl, or other substituted malonyl. Third, the loading modulespecificity also has an effect on the resulting carbon skeleton of thepolyketide. The loading module may use a different starter unit, such asacetyl, butyryl, and the like. As noted above, another method forvarying loading module specificity involves inactivating the KS activityin extender module 1 (KS1) and providing alternative substrates, calleddiketides, that are chemically synthesized analogs of extender module 1diketide products, for extender module 2. This approach was illustratedin PCT publication Nos. 97/02358 and 99/03986, incorporated herein byreference, wherein the KS1 activity was inactivated through mutation.Fourth, the oxidation state at various positions of the polyketide willbe determined by the dehydratase and reductase portions of the modules.This will determine the presence and location of ketone and alcoholmoieties and C—C double bonds or C—C single bonds in the polyketide.Finally, the stereochemistry of the resulting polyketide is a functionof three aspects of the synthase. The first aspect is related to theAT/KS specificity associated with substituted malonyls as extenderunits, which affects stereochemistry only when the reductive cycle ismissing or when it contains only a ketoreductase, as the dehydratasewould abolish chirality. Second, the specificity of the ketoreductasemay determine the chirality of any beta-OH. Finally, the enoylreductasespecificity for substituted malonyls as extender units may influence thestereochemistry when there is a complete KR/DH/ER available.

[0455] Thus, the modular PKS systems generally permit a wide range ofpolyketides to be synthesized. As compared to the aromatic PKS systems,the modular PKS systems accept a wider range of starter units, includingaliphatic monomers (acetyl, propionyl, butyryl, isovaleryl, etc.),aromatics (aminohydroxybenzoyl), alicyclics (cyclohexanoyl), andheterocyclics (thiazolyl). Certain modular PKSs have relaxed specificityfor their starter units (Kao et al., 1994, Science, supra). Modular PKSsalso exhibit considerable variety with regard to the choice of extenderunits in each condensation cycle. The degree of beta-ketoreductionfollowing a condensation reaction can be altered by genetic manipulation(Donadio et al., 1991, Science, supra; Donadio et al., 1993, Proc. Natl.Acad. Sci. USA 90: 7119-7123). Likewise, the size of the polyketideproduct can be varied by designing mutants with the appropriate numberof modules (Kao et al., 1994, J. Am. Chem. Soc. 116:11612-11613).Lastly, modular PKS enzymes are particularly well known for generatingan impressive range of asymmetric centers in their products in a highlycontrolled manner. The polyketides, antibiotics, and other compoundsproduced by the methods of the invention are typically singlestereoisomeric forms. Although the compounds of the invention can occuras mixtures of stereoisomers, it may be beneficial in some instances togenerate individual stereoisomers. Thus, the combinatorial potentialwithin modular PKS pathways based on any naturally occurring modular PKSscaffold is virtually unlimited. while hybrid PKSs are most oftenproduced by “mixing and matching” portions of PKS coding sequences,mutations in DNA encoding a PKS can also be used to introduce, alter, ordelete an activity in the encoded polypeptide. Mutations can be made tothe native sequences using conventional techniques. The substrates formutation can be an entire cluster of genes or only one or two of them;the substrate for mutation may also be portions of one or more of thesegenes. Techniques for mutation include preparing syntheticoligonucleotides including the mutations and inserting the mutatedsequence into the gene encoding a PKS subunit using restrictionendonuclease digestion. See, e.g., Kunkel, 1985, Proc. Natl. Acad. Sci.USA 82: 448; Geisselsoder et al., 1987, BioTechniques 5:786.Alternatively, the mutations can be effected using a mismatched primer(generally 10-20 nucleotides in length) that hybridizes to the nativenucleotide sequence, at a temperature below the melting temperature ofthe mismatched duplex. The primer can be made specific by keeping primerlength and base composition within relatively narrow limits and bykeeping the mutant base centrally located. See Zoller and Smith, 1983,Methods Enzymol. 100:468. Primer extension is effected using DNApolymerase, the product cloned, and clones containing the mutated DNA,derived by segregation of the primer extended strand, selected.Identification can be accomplished using the mutant primer as ahybridization probe. The technique is also applicable for generatingmultiple point mutations. See, e.g., Dalbie-McFarland et al., 1982,Proc. Natl. Acad. Sci. USA 79: 6409. PCR mutagenesis can also be used toeffect the desired mutations.

[0456] Random mutagenesis of selected portions of the nucleotidesequences encoding enzymatic activities can also be accomplished byseveral different techniques known in the art, e.g., by inserting anoligonucleotide linker randomly into a plasmid, by irradiation withX-rays or ultraviolet light, by incorporating incorrect nucleotidesduring in vitro DNA synthesis, by error-prone PCR mutagenesis, bypreparing synthetic mutants, or by damaging plasmid DNA in vitro withchemicals. Chemical mutagens include, for example, sodium bisulfite,nitrous acid, nitrosoguanidine, hydroxylamine, agents which damage orremove bases thereby preventing normal base-pairing such as hydrazine orformic acid, analogues of nucleotide precursors such as 5-bromouracil,2-aminopurine, or acridine intercalating agents such as proflavine,acriflavine, quinacrine, and the like. Generally, plasmid DNA or DNAfragments are treated with chemical mutagens, transformed into E. coliand propagated as a pool or library of mutant plasmids.

[0457] In constructing a hybrid PKS of the invention, regions encodingenzymatic activity, i.e., regions encoding corresponding activities fromdifferent PKS synthases or from different locations in the same PKS, canbe recovered, for example, using PCR techniques with appropriateprimers. By “corresponding” activity encoding regions is meant thoseregions encoding the same general type of activity. For example, a KRactivity encoded at one location of a gene cluster “corresponds” to a KRencoding activity in another location in the gene cluster or in adifferent gene cluster. Similarly, a complete reductase cycle could beconsidered corresponding. For example, KR/DH/ER can correspond to a KRalone.

[0458] If replacement of a particular target region in a host PKS is tobe made, this replacement can be conducted in vitro using suitablerestriction enzymes. The replacement can also be effected in vivo usingrecombinant techniques involving homologous sequences framing thereplacement gene in a donor plasmid and a receptor region in a recipientplasmid. Such systems, advantageously involving plasmids of differingtemperature sensitivities are described, for example, in PCT publicationNo. WO 96/40968, incorporated herein by reference. The vectors used toperform the various operations to replace the enzymatic activity in thehost PKS genes or to support mutations in these regions of the host PKSgenes can be chosen to contain control sequences operably linked to theresulting coding sequences in a manner such that expression of thecoding sequences can be effected in an appropriate host.

[0459] However, simple cloning vectors may be used as well. If thecloning vectors employed to obtain PKS genes encoding derived PKS lackcontrol sequences for expression operably linked to the encodingnucleotide sequences, the nucleotide sequences are inserted intoappropriate expression vectors. This need not be done individually, buta pool of isolated encoding nucleotide sequences can be inserted intoexpression vectors, the resulting vectors transformed or transfectedinto host cells, and the resulting cells plated out into individualcolonies. The invention provides a variety of recombinant DNA compoundsin which the various coding sequences for the domains and modules of thePKS are flanked by non-naturally occurring restriction enzymerecognition sites.

[0460] The various PKS nucleotide sequences can be cloned into one ormore recombinant vectors as individual cassettes, with separate controlelements, or under the control of, e.g., a single promoter. The PKSsubunit encoding regions can include flanking restriction sites to allowfor the easy deletion and insertion of other PKS subunit encodingsequences so that hybrid PKSs can be generated. The design of suchunique restriction sites is known to those of skill in the art and canbe accomplished using the techniques described above, such assite-directed mutagenesis and PCR.

[0461] The expression vectors containing nucleotide sequences encoding avariety of PKS enzymes for the production of different polyketides arethen transformed into the appropriate host cells to construct thelibrary. In one straightforward approach, a mixture of such vectors istransformed into the selected host cells and the resulting cells platedinto individual colonies and selected to identify successfultransformants. Each individual colony has the ability to produce aparticular PKS synthase and ultimately a particular polyketide.Typically, there will be duplications in some, most, or all of thecolonies; the subset of the transformed colonies that contains adifferent PKS in each member colony can be considered the library.Alternatively, the expression vectors can be used individually totransform hosts, which transformed hosts are then assembled into alibrary. A variety of strategies are available to obtain a multiplicityof colonies each containing a PKS gene cluster derived from thenaturally occurring host gene cluster so that each colony in the libraryproduces a different PKS and ultimately a different polyketide. Thenumber of different polyketides that are produced by the library istypically at least four, more typically at least ten, and preferably atleast 20, and more preferably at least 50, reflecting similar numbers ofdifferent altered PKS gene clusters and PKS gene products. The number ofmembers in the library is arbitrarily chosen; however, the degrees offreedom outlined above with respect to the variation of starter,extender units, stereochemistry, oxidation state, and chain lengthenables the production of quite large libraries.

[0462] Methods for introducing the recombinant vectors of the inventioninto suitable hosts are known to those of skill in the art and typicallyinclude the use of CaCl₂ or agents such as other divalent cations,lipofection, DMSO, protoplast transformation, infection, transfection,and electroporation. The polyketide producing colonies can be identifiedand isolated using known techniques and the produced polyketides furthercharacterized. The polyketides produced by these colonies can be usedcollectively in a panel to represent a library or may be assessedindividually for activity.

[0463] The libraries of the invention can thus be considered at fourlevels: (1) a multiplicity of colonies each with a different PKSencoding sequence; (2) the proteins produced from the coding sequences;(3) the polyketides produced from the proteins assembled into a functionPKS; and (4) antibiotics or compounds with other desired activitiesderived from the polyketides.

[0464] Colonies in the library are induced to produce the relevantsynthases and thus to produce the relevant polyketides to obtain alibrary of polyketides. The polyketides secreted into the media can bescreened for binding to desired targets, such as receptors, signalingproteins, and the like. The supernatants per se can be used forscreening, or partial or complete purification of the polyketides canfirst be effected. Typically, such screening methods involve detectingthe binding of each member of the library to receptor or other targetligand. Binding can be detected either directly or through a competitionassay. Means to screen such libraries for binding are well known in theart. Alternatively, individual polyketide members of the library can betested against a desired target. In this event, screens wherein thebiological response of the target is measured can more readily beincluded. Antibiotic activity can be verified using typical screeningassays such as those set forth in Lehrer et al., 1991, J. Immunol. Meth.137:167-173, incorporated herein by reference, and in the Examplesbelow.

[0465] The invention provides methods for the preparation of a largenumber of polyketides. These polyketides are useful intermediates information of compounds with antibiotic or other activity throughhydroxylation, epoxidation, and glycosylation reactions as describedabove. In general, the polyketide products of the PKS must be furthermodified, typically by hydroxylation and glycosylation, to exhibitantibiotic activity. Hydroxylation results in the novel polyketides ofthe invention that contain hydroxyl groups at C-6, which can beaccomplished using the hydroxylase encoded by the eryF gene, and/orC-12, which can be accomplished using the hydroxylase encoded by thepicK or eryK gene. Also, the oleP gene is available in recombinant form,which can be used to express the oleP gene product in any host cell. Ahost cell, such as a Streptomyces host cell or a Saccharopolysporaerythraea host cell, modified to express the oleP gene thus can be usedto produce polyketides comprising the C-8-C-8a epoxide present inoleandomycin. Thus the invention provides such modified polyketides. Thepresence of hydroxyl groups at these positions can enhance theantibiotic activity of the resulting compound relative to itsunhydroxylated counterpart.

[0466] Methods for glycosylating the polyketides are generally known inthe art; the glycosylation may be effected intracellularly by providingthe appropriate glycosylation enzymes or may be effected in vitro usingchemical synthetic means as described herein and in PCT publication No.WO 98/49315, incorporated herein by reference. Preferably, glycosylationwith desosamine, mycarose, and/or megosamine is effected in accordancewith the methods of the invention in recombinant host cells provided bythe invention. In general, the approaches to effecting glycosylationmirror those described above with respect to hydroxylation. The purifiedenzymes, isolated from native sources or recombinantly produced may beused in vitro. Alternatively and as noted, glycosylation may be effectedintracellularly using endogenous or recombinantly produced intracellularglycosylases. In addition, synthetic chemical methods may be employed.

[0467] The antibiotic modular polyketides may contain any of a number ofdifferent sugars, although D-desosamine, or a close analog thereof, ismost common. Erythromycin, picromycin, megalomicin, narbomycin, andmethymycin contain desosamine. Erythromycin also contains L-cladinose(3-O-methyl mycarose). Tylosin contains mycaminose (4-hydroxydesosamine), mycarose and 6-deoxy-D-allose. 2-acetyl-1-bromodesosaminehas been used as a donor to glycosylate polyketides by Masamune et al.,1975, J. Am. Chem. Soc. 97: 3512-3513. Other, apparently more stabledonors include glycosyl fluorides, thioglycosides, andtrichloroacetimidates; see Woodward et al., 1981, J. Am. Chem. Soc. 103:3215; Martin et al., 1997, J. Am. Chem. Soc. 119: 3193; Toshima et al.,1995, J. Am. Chem. Soc. 117: 3717; Matsumoto et al., 1988, TetrahedronLett. 29: 3575. Glycosylation can also be effected using the polyketideaglycones as starting materials and using Saccharopolyspora erythraea orStreptomyces venezuelae or other host cell to make the conversion,preferably using mutants unable to synthesize macrolides, as discussedabove.

[0468] Thus, a wide variety of polyketides can be produced by the hybridPKS enzymes of the invention. These polyketides are useful asantibiotics and as intermediates in the synthesis of other usefulcompounds. In one important aspect, the invention provides methods formaking antibiotic compounds related in structure to erythromycin, apotent antibiotic compound. The invention also provides novel ketolidecompounds, polyketide compounds with potent antibiotic activity ofsignificant interest due to activity against antibiotic resistantstrains of bacteria. See Griesgraber et al., 1996, J. Antibiot. 49:465-477, incorporated herein by reference. Most if not all of theketolides prepared to date are synthesized using erythromycin A, aderivative of 6-dEB, as an intermediate. See Griesgraber et al., supra;Agouridas et al., 1998, J. Med. Chem. 41: 4080-4100, U.S. Pat. Nos.5,770,579; 5,760,233; 5,750,510; 5,747,467; 5,747,466; 5,656,607;5,635,485; 5,614,614; 5,556,118; 5,543,400; 5,527,780; 5,444,051;5,439,890; 5,439,889; and PCT publication Nos. WO 98/09978 and 98/28316,each of which is incorporated herein by reference.

[0469] As noted above, the hybrid PKS genes of the invention can beexpressed in a host cell that contains the desosamine, megosamine,and/or mycarose biosynthetic genes and corresponding transferase genesas well as the required hydroxylase gene(s), which may be either picK,megK, or eryK (for the C-12 position) and/or megF oreryF (for the C-6position). The resulting compounds have antibiotic activity but can befurther modified, as described in the patent publications referencedabove, to yield a desired compound with improved or otherwise desiredproperties. Alternatively, the aglycone compounds can be produced in therecombinant host cell, and the desired glycosylation and hydroxylationsteps carried out in vitro or in vivo, in the latter case by supplyingthe converting cell with the aglycone, as described above.

[0470] As described above, there are a wide variety of diverse organismsthat can modify compounds such as those described herein to providecompounds with or that can be readily modified to have usefulactivities. For example, Saccharopolyspora erythraea can convert 6-dEBto a variety of useful compounds. The compounds provided by the presentinvention can be provided to cultures of Saccharopolyspora erythraea andconverted to the corresponding derivatives of erythromycins A, B, C, andD in accordance with the procedure provided in the Examples, below. Toensure that only the desired compound is produced, one can use an S.erythraea eryA mutant that is unable to produce 6-dEB but can stillcarry out the desired conversions (Weber et al., 1985, J. Bacteriol.164(1): 425-433). Also, one can employ other mutant strains, such aseryB, eryC, eryG, and/or eryK mutants, or mutant strains havingmutations in multiple genes, to accumulate a preferred compound. Theconversion can also be carried out in large fermentors for commercialproduction. Each of the erythromycins A, B, C, and D has antibioticactivity, although erythromycin A has the highest antibiotic activity.Moreover, each of these compounds can form, under treatment with mildacid, a C-6 to C-9 hemiketal with motilide activity. For formation ofhemiketals with motilide activity, erythromycins B, C, and D, arepreferred, as the presence of a C-12 hydroxyl allows the formation of aninactive compound that has a hemiketal formed between C-9 and C-12.

[0471] Thus, the present invention provides the compounds produced byhydroxylation and glycosylation of the compounds of the invention byaction of the enzymes endogenous to Saccharopolyspora erythraea andmutant strains of S. erythraea. Such compounds are useful as antibioticsor as motilides directly or after chemical modification. For use asantibiotics, the compounds of the invention can be used directly withoutfurther chemical modification. Erythromycins A, B, C, and D all haveantibiotic activity, and the corresponding compounds of the inventionthat result from the compounds being modified by Saccharopolysporaerythraea also have antibiotic activity. These compounds can bechemically modified, however, to provide other compounds of theinvention with potent antibiotic activity. For example, alkylation oferythromycin at the C-6 hydroxyl can be used to produce potentantibiotics (clarithromycin is C-6-O-methyl), and other usefulmodifications are described in, for example, Griesgraber et al., 1996,J. Antibiot. 49: 465-477, Agouridas et al., 1998, J. Med. Chem. 41:4080-4100, U.S. Pat. Nos. 5,770,579; 5,760,233; 5,750,510; 5,747,467;5,747,466; 5,656,607; 5,635,485; 5,614,614; 5,556,118; 5,543,400;5,527,780; 5,444,051; 5,439,890; and 5,439,889; and PCT publication Nos.WO 98/09978 and 98/28316, each of which is incorporated herein byreference.

[0472] For use as motilides, the compounds of the invention can be useddirectly without further chemical modification. Erythromycin and certainerythromycin analogs are potent agonists of the motilin receptor thatcan be used clinically as prokinetic agents to induce phase III ofmigrating motor complexes, to increase esophageal peristalsis and LESpressure in patients with GERD, to accelerate gastric emptying inpatients with gastric paresis, and to stimulate gall bladdercontractions in patients after gallstone removal and in diabetics withautonomic neuropathy. See Peeters, 1999, Motilide Web Site,http://www.med.kuleuven. ac.be/med/gih/motilid.htm, and Omura et al.,1987, Macrolides with gastrointestinal motor stimulating activity, J.Med. Chem. 30: 1941-3). The corresponding compounds of the inventionthat result from the compounds of the invention being modified bySaccharopolyspora erythraea also have motilide activity, particularlyafter conversion, which can also occur in vivo, to the C-6 to C-9hemiketal by treatment with mild acid. Compounds lacking the C-12hydroxyl are especially preferred for use as motilin agonists. Thesecompounds can also be further chemically modified, however, to provideother compounds of the invention with potent motilide activity.

[0473] Moreover, and also as noted above, there are other usefulorganisms that can be employed to hydroxylate and/or glycosylate thecompounds of the invention. As described above, the organisms can bemutants unable to produce the polyketide normally produced in thatorganism, the fermentation can be carried out on plates or in largefermentors, and the compounds produced can be chemically altered afterfermentation. In addition to Saccharopolyspora erythraea, Streptomycesvenezuelae, S. narbonensis, S. antibioticus, Micromonospora megalomicea,S. fradiae, and S. thermotolerans can also be used. In addition toantibiotic activity, compounds of the invention produced by treatmentwith M. megalomicea enzymes can have antiparasitic activity as well.Thus, the present invention provides the compounds produced byhydroxylation and glycosylation by action of the enzymes endogenous toS. erythraea, S. venezuelae, S. narbonensis, S. antibioticus, M.megalomicea, S. fradiae, and S. thermotolerans.

[0474] The compounds of the invention can be isolated from thefermentation broths of these cultured cells and purified by standardprocedures. The compounds can be readily formulated to provide thepharmaceutical compositions of the invention. The pharmaceuticalcompositions of the invention can be used in the form of apharmaceutical preparation, for example, in solid, semisolid, or liquidform. This preparation will contain one or more of the compounds of theinvention as an active ingredient in admixture with an organic orinorganic carrier or excipient suitable for external, enteral, orparenteral application. The active ingredient may be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, suppositories, solutions, emulsions,suspensions, and any other form suitable for use.

[0475] The carriers which can be used include water, glucose, lactose,gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate,talc, corn starch, keratin, colloidal silica, potato starch, urea, andother carriers suitable for use in manufacturing preparations, in solid,semi-solid, or liquified form. In addition, auxiliary stabilizing,thickening, and coloring agents and perfumes may be used. For example,the compounds of the invention may be utilized with hydroxypropylmethylcellulose essentially as described in U.S. Pat. No. 4,916,138,incorporated herein by reference, or with a surfactant essentially asdescribed in EPO patent publication No. 428,169, incorporated herein byreference.

[0476] Oral dosage forms may be prepared essentially as described byHondo et al., 1987, Transplantation Proceedings XIX, Supp. 6: 17-22,incorporated herein by reference. Dosage forms for external applicationmay be prepared essentially as described in EPO patent publication No.423,714, incorporated herein by reference. The active compound isincluded in the pharmaceutical composition in an amount sufficient toproduce the desired effect upon the disease process or condition.

[0477] For the treatment of conditions and diseases caused by infection,a compound of the invention may be administered orally, topically,parenterally, by inhalation spray, or rectally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvant, and vehicles. The term parenteral, asused herein, includes subcutaneous injections, and intravenous,intramuscular, and intrasternal injection or infusion techniques.

[0478] Dosage levels of the compounds of the invention are of the orderfrom about 0.01 mg to about 50 mg per kilogram of body weight per day,preferably from about 0.1 mg to about 10 mg per kilogram of body weightper day. The dosage levels are useful in the treatment of theabove-indicated conditions (from about 0.7 mg to about 3.5 mg perpatient per day, assuming a 70 kg patient). In addition, the compoundsof the invention may be administered on an intermittent basis, i.e., atsemi-weekly, weekly, semi-monthly, or monthly intervals.

[0479] The amount of active ingredient that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. Forexample, a formulation intended for oral administration to humans maycontain from 0.5 mg to 5 gm of active agent compounded with anappropriate and convenient amount of carrier material, which may varyfrom about 5 percent to about 95 percent of the total composition.Dosage unit forms will generally contain from about 0.5 mg to about 500mg of active ingredient. For external administration, the compounds ofthe invention may be formulated within the range of, for example,0.00001% to 60% by weight, preferably from 0.001% to 10% by weight, andmost preferably from about 0.005% to 0.8% by weight.

[0480] It will be understood, however, that the specific dose level forany particlular patient will depend on a variety of factors. Thesefactors include the activity of the specific compound employed; the age,body weight, general health, sex, and diet of the subject; the time androute of administration and the rate of excretion of the drug; whether adrug combination is employed in the treatment; and the severity of theparticular disease or condition for which therapy is sought.

[0481] A detailed description of the invention having been providedabove, the following examples are given for the purpose of illustratingthe invention and shall not be construed as being a limitation on thescope of the invention or claims.

EXAMPLE 1 Production of Methylmalonyl-CoA in E. Coli

[0482] This example describes, in part A, the cloning and expression ofmethylmalonyl-CoA mutase, and in part B, the cloning and expression ofmethylmalonyl-CoA epimerase, in E. coli.

[0483] A. Cloning and Expression of Methylmalonyl-CoA Mutase

[0484] Methylmalonyl-CoA mutase was cloned from Propionibacteriumshermanii and expressed in E. coli. The holoenzyme mm-CoA mutase wasobtained by growing cells in the presence of hydroxocobalamin and wasshown to be active without addition of vitamin B12. Methylmalonyl-CoAwas produced in vivo, as seen by CoA analysis using a panD strain ofBL21 (DE3).

[0485] To support modular polyketide production in E. coli, theinvention provides methods and reagents to produce(S)-methylmalonyl-CoA, which is not naturally present in E. coli, byoverexpressing mm-CoA mutase and mm-CoA epimerase in E. coli. An active,FLAG-tagged version of the mm-CoA mutase from S. cinnamonensis wasexpressed in XL1Blue cells, which were grown in the presence ofhydroxocobalamin in a synthetic, vitamin-free media to produce activeholoenzyme. The CoA levels in the cells were analyzed by feeding labeledβ-alanine; for this purpose it is beneficial to have a panD strain,which is a β-alanine auxotroph. The mutase DNA rearranged in the panDstrain of SJ16, a recA⁺ strain, such that the CoA analysis had to becarried out without the panD. This resulted in a lower signal to noiseratio, but elevated mm-CoA levels could still be detected. As analternative to the S. cinnamonensis genes, the invention provides amm-CoA mutase from P. shermanii cloned into an E. coli expressionvector, which is active without addition of vitamin B12, and whichelevates mm-CoA levels in E. coli in a panD strain compatible with themutase DNA.

[0486]Propionibacterium freudenreichii subsp. shermanii was obtained asa stab in tomato juice agar from derived from a freeze-dried specimenfrom NCIMB, Scotland (NCIMB # 9885). E. coli strain gg3, a panD versionof BL21 (DE3) was used for the CoA analysis. E. coli strains gg1 andgg2, recA⁻ versions of the SJ16 panD strain, were also used. The vectorpKK** is a version of pKK223-3 in which the cloning region is altered torange from Nde1 to EcoR1 and an extra Nde1 site is deleted. Growth of P.shermanii and preparation of genomic DNA was conducted as described inthe literature.

[0487] Subcloning of methylmalonyl-CoA mutase from P. shermanii into E.coli was conducted as follows. The gene for mm-CoA mutase consists oftwo subunits, mutA and mutB, which were amplified by PCR from P.shermanii genomic DNA in a total of four fragments. Naturally occurringrestriction sites were used to piece the gene together. Uniquerestriction sites were introduced at both ends of the gene for cloningpurposes, and the start codon for the mutB gene was changed from GTG toATG. As illustrated below, these four fragments were cloned into aBluescript™ (Stratagene) vector, sequenced, and then pieced together toform the complete mutase gene. The gene was then cloned into expressionvectors pET22b and pKK** between the restriction sites Ndel and HindIII,to form pET-MUT and pKK**-MUT.

[0488] The pET-MUT was transformed into competent cells BL21(DE3) andlater into cells gg3, which are a panD version of BL21(DE3). ThepKK**-MUT was transformed into SJ16 panD and into XL1Blue. The DNA wastested by screening several colonies with Ndel and HindIII, to determineif the mutase gene was still present or if it had rearranged.

[0489] For SDS-PAGE analysis, cells of strain BL21(DE3) containingpET-MUT (and pET alone, as a control) were grown aerobically at 27° C.in MUT media with 100 μg/ml carbenicillin (carb) (MUT media is M9 salts,glucose, thiamine, trace elements and amino acids, as previouslydescribed for the expression of methionine synthase (Amaratunga, 1996)).Overnight cultures (250 μl) were used to inoculate 25 mL of MUT media(carb), which were grown at 27° C. to an OD₆₀₀ of approximately 0.5. Thecultures were then induced with IPTG to 1 mM final concentration. Twocultures were left at 27° C. for three hours while duplicate cultureswere grown at 37° C. for two hours. The cells were collected bycentrifugation and the pellets were stored at −80° C. prior to analysis.The cells were lysed by sonication and both the soluble and insolublephases were examined by SDS/PAGE. This procedure was repeated for cellsof strain XL1Blue containing pKK**-MUT.

[0490] For expression of active mm-CoA mutase (with hydroxocobalamin),cells of strain gg3 containing pET-MUT (and pET alone, as a control)were grown in MUT media (carb) and 5 EM beta-alanine for approximately20 hours at 27° C. The following operations were performed in a darkroom with a red safelight: 125-mL flasks, each containing 25 mL of MUTmedia with carb and 5 μM β-alanine and wrapped in aluminum foil, wereinoculated with 5 μM hydroxocobalamin and then with 250 μL from therespective starter cultures. After shaking overnight at 27° C., thecultures were induced with IPTG to 1 mM final concentration and grownfor an additional 4:45 hours, at which point they were collected (inFalcon tubes wrapped in aluminum foil) by centrifugation at 4000 rpm for10 minutes. The pellets were stored in the dark at −80° C. prior toassaying.

[0491] The mutase assay was performed as follows. All operations wereperformed in the dark or under a red safelight. The pellet from 25 mL ofculture was thawed, washed in buffer C (50 mM potassium phosphate pH7.4, 5 mM EDTA, 10% glycerol), and resuspended in 0.5 mL of buffer Ccontaining protease inhibitors (1 tablet per 10 mL of buffer). Followingsonication on ice, the extract was clarified by centrifugation at 4° C.for 10 minutes at maximum speed in an Eppendorf microfuge; thesupernatent was assayed. Enzyme assays contained, in a final volume of100 μL, 0.2 mM (2R,2S)-methylmalonyl-CoA, mutase extract, and buffer Ccontaining protease inhibitors. Reactions for assays with vitamin B12were as above but contained 0.01 mM vitamin B12, in which case themutase extract was incubated with the vitamin B12 in a total volume of75 μL for 5 minutes at 30° C. prior to initiation of reaction withmethylmalonyl-CoA. After the desired length of incubation at 30° C., thereaction was stopped by the addition of 50 μL of 10% trichloroaceticacid (TCA) and placed on ice for approximately 10 minutes. Cellulardebris and precipitated protein were removed by centrifugation for 5minutes in an Eppendorf microfuge at 4° C. An aliquot (100 μL) of thesupernatant was injected onto the HPLC to quantify conversion ofmethylmalonyl-CoA to succinyl-CoA. One time point was taken after 20minutes of incubation at 30° C., and the sample was assayed forconversion of mm-CoA to succinyl-CoA. All operations were performedexclusively under a red safelight until the reaction was stopped byaddition of TCA.

[0492] The CoA analysis was performed as described in the literature,except that 5 μM of hydroxocobalamin were added at the time of IPTGinduction, and the tubes were wrapped in aluminum foil and grown at 27°C. instead of 30° C. The CoA peaks, which eluted in approximately oneminute each, were collected manually, as well as approximately oneminute of sample both before and after each peak. In some tests,fractions were collected every 30 seconds. All samples were counted inthe scintillation counter.

[0493] The two subunits of the gene encoding methylmalonyl-CoA mutaseare translationally coupled—the GTG start codon of the downstreamsubunit mutB overlaps with the ATG codon of mutA. The GTG valine startwas mutated to an ATG methionine start (which does not alter any otheramino acids), because E. coli utilizes the methionine start moreefficiently. Sequencing the mm-CoA mutase gene revealed a discrepancybetween the sequence observed and the published sequence (117-7). A “GC”instead of a “CG” changed two amino acids from Asp, Val to Glu, Leu. Thecrystal structure of mm-CoA mutase from P. shermanii (1996) showed thatthe two amino acids are indeed Glu, Leu, so the published sequence is inerror. The mm-CoA mutase gene was subcloned into two different E. coliexpression systems: pET, which is under control of the strong T7promoter, and pKK, which uses the leaky tac promoter. First it wasnecessary to find strains in which the mutase DNA did not rearrange. Itwas previously observed that a FLAG-tagged version of the mutase from S.cinnamonensis rearranged in SJ16 panD and in BL21(DE3), which are bothrecA⁺ strains, but not in XL1Blue, which is recA⁻. This mutase DNA (P.shermanii) also rearranged in the SJ16 cells but not in the BL21(DE3)cells. Thus a panD version of BL21(DE3) was created (gg3) for use withthe pET vector. A recA⁻ version of SJ16 was also created (gg1, gg2) foruse with the pKK system; however, the mutase DNA rearranged in thisstrain as well.

[0494] Different growth conditions were tested to find conditions inwhich the two subunits of the mutase were expressed in the soluble phasein approximately equal molar ratios. In general, it seemed that thehigher temperature of 37° C. caused the mutase to appear predominantlyin the insoluble form. Growth exclusively at 27° C. resulted in solubleprotein with an approximately equal subunit ratio.

[0495] The graph below shows the comparison of in vivo acyl-CoA levelsin BL21(DE3) panDstrains with and without mm-CoA mutase. For each CoA,the ratio of the amount in the strain containing the mutase to theamount in the control strain was determined. Interestingly, malonyl-CoAwas increased about 25-fold and succinyl-CoA about 3-fold. Acetyl-CoAand CoA were increased just slightly, and propionyl-CoA was not detectedin either case.

[0496] To express active mutase in vivo, it was necessary to grow cellsin a defined media (MUT media) that allows uptake of the vitamin B12precursor hydroxocobalamin; this is similar to an established protocolfor expression of active methionine synthase, which also requires B12.Cell extracts overexpressing the mutase were shown to convert mm-CoA tosuccinyl CoA without the addition of vitamin B12. Only one time point(at 20 minutes) was assayed to confirm activity; the specific activityof the mutase must was not determined.

[0497] Thus, methylmalonyl-CoA mutase was expressed as the activeholoenzyme in E. coli, and methylmalonyl-CoA was produced in vivo.Because a slow, spontaneous chemical epimerization between (R)- and(S)-mm-CoA does exist (approximately 3% in 15 minutes), it may behelpfuL to determine the relative amounts of these diastereomers incells overexpressing the mutase. Enough (S)-mm-CoA may be present tosupport polyketide production in some cells without addition of anepimerase. To facilitate the eventual production of polyketides in E.coli, the mutase gene can be incorporated into the chromosome of theBL21 panD cell or other host cell.

[0498] The schematic below shows the construction of pSK-MUT, in whichfour PCR fragments were sequenced and pieced together to form thecomplete mutase gene in pSK-bluescript.

[0499]

[0500] In follow-up experiments, the specific activity of the mutase wasdetermined and an in-depth CoA analysis was completed. The CoA levels inthe cells were again analyzed using a pand strain, which is a β-alanineauxotroph. ³H-β-alanine was fed to the cells and incorporated into theacyl-CoAs, which were separated via HPLC and counted. The CoA pools forcell extracts with and without the mutase, as well as with and withouthydroxocobalamin, were examined.

[0501] To test whether acyl-CoAs degrade in TCA, the following testswere conducted. The CoA mix consisted of 1.6 mM each of malonyl-,methylmalonyl-, succinyl-, acetyl-, and propionyl-CoA, plus 0.5 mM CoA.An aliquot (10 μL) of this mix was added to 100 μL 10% TCA, 50 μL wereimmediately injected to the HPLC for CoA analysis, and the remainder waspromptly frozen on dry ice. The frozen portion was then thawed andloaded immediately to the HPLC. Again, 10 μL of the CoA mix were addedto 100 μL 10% TCA, 50 μL were left on ice for 15 minutes and theninjected to the HPLC, the remainder was left at 4° C. overnight andinjected to the HPLC the next morning. The area under each CoA peak wasnoted. This same procedure was followed but using a mixture of TCA andbuffer A from the mutase assay.

[0502] The CoA analysis described here is carried out on cells which arelysed in 10% TCA. Thus, determining whether the CoAs degradesignificantly in TCA and in a mixture of TCA and buffer A from themutase assay is important. The tests showed that the percent of each CoArelative to the total CoA pool, as well as the overall amount of CoA,remained constant after freeze/thawing, after leaving on ice for 15minutes, and after leaving the sample overnight at 4° C. Thus, the CoAsare stable in TCA and in the mutase assay buffer after the cells arelysed or after the assays are completed, and prior to HPLC analysis.

[0503] Although the CoAs are stable in TCA and buffer at 4° C., theydegraded at 30° C., the temperature at which the mutase assay wasperformed. In five minutes under the assay conditions, about 4% of themethylmalonyl-CoA hydrolyzed to CoA. The succinyl-CoA hydrolyzed at acomparable rate. Thus, the mutase assay is suboptimal for extremelyquantitative results.

[0504] When 0.2 mM methylmalonyl-CoA was incubated with a crude lysatefrom cell extracts overexpressing the mutase, succinyl-CoA was produced.No succinyl-CoA was observed when methylmalonyl-CoA was incubated withlysates from the control strain (containing the plasmid vector butlacking the mutase genes). Under these expression and assay conditions,a specific activity of approximately 0.04 U/mg was observed in the crudeextracts. When cells overexpressing the mutase were grown in MUT mediawithout hydroxocobalamin, no mutase activity was observed; however,mutase activity could be detected by addition of vitamin B12 in vitro.Adding vitamin B12 to extracts that were grown in the presence ofhydroxocobalamin resulted in increased mutase activity, suggesting thata significant amount of expressed mutase is present as the apo-enzyme.This might have occurred because the enzyme was expressed faster thanthe hydroxocobalamin could be transported into the cell, or because thevitamin B12 cofactor was lost during preparation of the extract.

[0505] The graph below shows the comparison of in vivo acyl-CoA levelswith and without the mutase and with and without hydroxocobalamin. Inthe cells overexpressing the mutase and grown with hydroxocobalamin,methylmalonyl-CoA comprised 13% of the overall CoA pool, whereas in theother cells no methylmalonyl-CoA was detectable. The background level ofcounts is about 0.25% of the overall number of counts in the CoAs,suggesting that any methymalonyl-CoA present in E. coli strains notoverexpressing the mutase would comprise at most 0.25% of the overallCoA pool, or 2% of the amount of methylmalonyl-CoA observed in thestrain overexpressing the mutase. The composition of the CoA poolobserved for the E. coli panD strain is consistent with that observedpreviously for E. coli panD mutants grown on glucose.

[0506] Thus, the methylmalonyl-CoA mutase from P. shermanii has beenoverexpressed as the active holoenzyme in E. coli and shown to produce(2R)-methylmalonyl-CoA in vivo. Conversion of (2R)- to(2S)-methylmalonyl-CoA via methylmalonyl-CoA epimerase should provide anadequate supply of the correct isomer of methylmalonyl-CoA to supportheterologous production of complex polyketides E. coli.

[0507] The graph above shows the results of CoA analysis of E. colioverexpressing methylmalonyl-CoA mutase. The levels of ³H detected infractions collected from HPLC of cell-free extracts from ³Hβ-alanine-fed E. coli harboring either the pET control vector grownwithout hydroxocobalamin (black trace), pET grown with hydroxocobalamin(blue trace), pET overexpressing the mutase and grown withouthydroxocobalamin (green trace), or pET overexpressing the mutase andgrown with hydroxocobalamin (red trace) are shown.

[0508] B. Cloning and Expression of Methylmalonyl-CoA Epimerase

[0509] The mm-CoA epimerase from Propionibacterium shermanii waspurified and used to obtain N-terminal protein sequence as well asinternal peptide sequence from LysC-generated peptides. The epimerasegene was cloned using hybridization probes designed from the peptidesequences.

[0510]Propionibacterium freudenreichii subsp. shermanii was obtained andcultured as described in part A. Purification of mm-CoA epimerase fromP. shermanii was based on a modification of the published procedure. Theprocedure utilized a 10 L culture, which was lysed by sonicationfollowed by column chromatography in the order: DE-52, Hydroxyapatite,Phenylsepharose, MonoQ anion exchange, and C-8 RP HPLC.

[0511] All operations were performed at 4° C., except the C-8 RP HP2C,which was performed at room temperature, and all buffers contained 0.1mM PMSF, unless otherwise stated. The epimerase assay was performedessentially as described in the literature. Protein concentration wasdetermined using the method of Bradford. The overall yield of epimeraseactivity was not determined.

[0512] More specifically, cell paste (75 g) was resuspended in 50 mLbuffer (5 mM Tris-HCl pH 7.5, 0.1 M KCl, 0.2 mM PMSF, 1 mM EDTA) andsonicated using a macrotip with a diameter of 1.2 cm. With pulses of 0.5seconds ON and 0.3 seconds OFF, the cells were sonicated twice for 30seconds each at a power setting of 4, followed by five times for 30seconds each at a power setting of 6. A clear, amber-colored supernatant(53.5 ml) was obtained after spinning for 35 minutes at 12,000 rpm.

[0513] The crude extract from above was applied to a column (diameter2.5 cm, height 15 cm) of 73 mL of DE-52 resin equilibrated with 50 mMTris-HCl pH 7.5, 0.1 M KCl. The column was washed at 1 ml/min with threecolumn volumes of the above buffer, followed by a linear gradient to 5mM Tris-HCl pH 7.5, 0.5 M KCl over seven column volumes. Six mLfractions were collected and assayed for epimerase activity. Theepimerase was found predominantly in the flow-through and in severalearly fractions. The flow-through and active fractions were combined(325 mL) and dialyzed against 4 liters of 50 mM Tris-HCl pH 7.5, 10%glycerol, followed by 4 liters of 10 mM sodium phosphate pH 6.5, 10%glycerol (final volume 250 mL).

[0514] A 7.5 mL hydroxyapatite biogel HTP gel column (diameter 1.5 cm,height 16 cm) was equilibrated with 10 mM sodium phosphate pH 6.5, 5%glycerol. After loading of the enzyme solution (using repeatedinjections) and washing with three column volumes of the above buffer, agradient to 200 mM sodium phosphate pH 6.5, 5% glycerol was effectedover 20 column volumes at a flow rate of 1 ml/min. The 2 mL fractionswere assayed for epimerase activity, and fractions containing epimeraseactivity were pooled for a total of 99 ml.

[0515] To the 99 mL sample from above, solid ammonium sulfate to 1.5 Mfinal concentration was added slowly and with stirring at 4° C. over 30minutes. This suspension (100 mL) was loaded, by repeated injection,onto a 6.6 mL column (1 cm×height 8.5 cm) of phenyl-sepharose resinequilibrated in 20 mM sodium phosphate buffer pH 6.5, 1.5 M ammoniumsulfate. The column was washed at 1 ml/min with three column volumes ofthis buffer, followed by a linear gradient to 20 mM sodium phosphatebuffer pH 6.5, 10% glycerol, over 24 column volumes. After assaying the3 mL fractions for epimerase activity, the fractions containingepimerase activity were pooled and dialyzed against 5 mM Tris-HCl pH7.5.

[0516] A mono Q 5/5 prepacked column was equilibrated with 25 mMTris-HCl pH 7.5 at 0.5 mL/min. The sample from the previous step wasloaded onto the column, which was then washed with 5 column volumes ofthe above buffer, followed by a linear gradient to 50 mM Tris-HCl pH7.5, 1 M NaCl, 5% glycerol, over 50 column volumes. The 1 mL fractionswere assayed for epimerase activity. Several fractions containingepimerase activity were stored separately; the fraction with the mostactivity was used for the next purification step.

[0517] A reverse-phase column was equilibrated with water containing0.1% trifluoroacetic acid; 120 μL (concentrated from 0.5 mL of theactive fraction from above, using an Amicon microconcentrator) wasinjected onto the column at a flow rate of 0.2 mL/min and washed forfive minutes with the above solvent system. Then a linear gradient over50 minutes to acetonitrile containing 0.1% trifluoroacetic acid wasimplemented. The peaks were collected manually and the peakcorresponding to the epimerase (as determined by SDS/PAGE) was dried tocompleteness, resuspended in water and stored at −80° C.

[0518] For Lys C mediated digestion of the HPLC-purified epimerase, theepimerase fraction (11751rp2-B, 200 μL) collected from reverse phaseHPLC was dried to completeness and resuspended in 40 μL water. To 30 μLof the sample was added 5 μL of 1 M Tris/HCl, pH 8, 1.5 μL of 0.1 M DTT,2 μL of Lys C protease (0.2 μg). A control reaction contained all of theabove components except the epimerase. The reactions were incubatedovernight at 37° C. An aliquot of the reaction (5 μL) was diluted to 60μL with water and loaded to the HPLC, using the same HPLC program thatwas used to purify the epimerase. The analytical HPLC showed that theLys C digestion was not complete. An additional aliquot of Lys C (0.2μg) was added to the reactions and incubation was continued overnight at37° C. Following overnight incubation, an aliquot of the reaction (5 μL)was diluted to 60 μL with water and subjected to the HPLC. The HPLCshowed that the digestion was complete. The remainder of the reactionwas loaded to the HPLC and individual peaks were collected manually.HPLC of the control reaction showed no significant peptide fragmentsarising from self-digestion of the LysC.

[0519] An aliquot of the pure epimerase, as well as a peptide collectedfrom the procedure described above, were submitted for N-terminal aminoacid sequencing. Based on the amino acid sequences from above, severaldegenerate primers were designed as described below that introducedunique restriction sites to either end of the eventual PCR product.These primers were used in PCR with P. shermanii genomic DNA to obtain a200 base-pair product, which was cloned into a Bluescript™ (Stratagene)vector and submitted for sequencing.

[0520] A cosmid library of P. shermanii was prepared, essentially asdescribed in the Stratagene cosmid manual. The titer of this cosmidlibrary was approximately 11 cfu (colony forming units) per μL, for atotal yield of 5556 cfu. A plasmid library of P. shermanii was preparedby digesting P. shermanii genomic DNA with SacI and ligating theresulting mixture into a Bluescript™ vector also cut with SacI. Todetermine the average insert size (2 kb), ten random clones weredigested with SacI. The ligation mixture was re-transformed 5 times,pooled and plated on one large LB (carb) plate, resulting in a lawn ofcolonies that were scraped together and resuspended in LB as the plasmidlibrary. The titer of this plasmid library was approximately 64,000 cfuper μL.

[0521] Several degenerate primers based on the amino acid sequences wereprepared and used in PCR with P. shermanii genomic DNA to obtain a 180base-pair product, which was cloned into a Bluescript™ vector andsequenced. Several different probes were made. The first probe was madeusing the random priming method to incorporate either ³²P or digoxigenininto the epimerase fragment. A probe was made from the cloned fragmentby amplification of the fragment via PCR, using the digoxigenin labelingmethod. The PCR product was gel isolated, quantified, and used to probethe cosmid library. Colonies that hybridized to the probe wererestreaked from master plates, and five colonies from the re-streakedplates were picked, cosmids were isolated, and the insert sequencesscreened for the epimerase gene by PCR. Several cosmids that were scoredpositive for epimerase DNA sequence by PCR were subjected to DNAsequencing using epimerase-specific primers. The cosmid designated117-167-A7 contained the full epimerase sequence.

[0522] The sequence of the putative epimerase gene contained in cosmid117-167-A7 was aligned to the N-terminal epimerase sequence alreadyknown. The several hundred base pairs downstream of this sequence weretranslated in all three frames and a stop codon in one of the frames wasfound that yielded a protein of the expected size. The entire sequencewas used to search the protein database via BLAST analysis, and thesequence showed high homology to the sequence of a putative epimerasefrom S. coelicolor identified in accordance with the methods of theinvention. PCR primers were designed based on the DNA sequence of thecloned P. shermanii epimerase and the gene was amplified from P.shermanii genomic DNA with NdeI and BamHI sites at the 5′-end, aninternal NdeI site was destroyed near the 5′ end, and NheI and AvrIIsites were introduced at the 3′-end. Following PCR, the 447 bp productwas cloned into a Bluescript vector (143-6-11) and sequenced. Also, fouradditional sequencing primers were designed to provide several-foldcoverage of the epimerase gene. The full epimerase gene sequenceprovided in isolated and recombinant form by the present invention isshown below.                                                 50ATGAGTAATGAGGATCTTTTCATCTGTATCGATCACGTGGCATATGCGTG M  S  N  E  D  L  F  I  C  I  D  H  V  A  Y  A  C                                               100CCCCGACGCCGACGAGGCTTCCAAGTACTACCAGGAGACCTTCGGCTGGC  P  D  A  D  E  A  S  K  Y  Y  Q  E  T  F  G  W                                               150ATGAGCTCCACCGCGAGGAGAACCCGGAGCAGGGAGTCGTCGAGATCATGH  E  L  H  R  E  E  N  P  E  Q  G  V  V  E  I  M                                               200ATGGCCCCGGCTGCGAAGCTGACCGAGCACATGACCCAGGTTCAGGTCAT M  A  P  A  A  K  L  T  E  H  M  T  Q  V  Q  V  M                                               250GGCCCCGCTCAACGACGAGTCGACCGTTGCCAAGTGGCTTGCCAAGCACA  A  P  L  N  D  E  S  T  V  A  K  W  L  A  K  H                                               300ATGGTCGCGCCGGACTGCACCACATGGCATGGCGTGTCGATGACATCGACN  G  R  A  G  L  H  H  M  A  W  R  V  D  D  I  D                                               350GCCGTCAGCGCCACCCTGCGCGAGCGCGGCGTGCAGCTGCTGTATGACGA A  V  S  A  T  L  R  E  R  G  V  Q  L  L  Y  D  E                                               400GCCCAAGCTCGGCACCGGCGGCAACCGCATCAACTTCATGCATCCCAAGT  P  K  L  G  T  G  G  N  R  I  N  F  M  H  P  KCGGGCAAGGGCGTGCTCATCGAGCTCACCCAGTACCCGAAGAACTGAS  G  K  G  V  L  I  E  L  T  Q  Y  P  K  N  *

[0523] The epimerase gene was then cloned into a pET expression vector;the construct was named pET-epsherm.

[0524] For the cloning of epimerase genes from B. subtilis (described byHaller et al., supra) and S. coelicolor (from cosmid 8F4 in the S.coelicolor, genome sequencing project), primers were designed to PCRthese genes from their respective genomic DNAs and to incorporate eithera PacI or NdeI site at the 5′ end, and an NsiI site at the 3′ end. ThePCR products were cloned into a Bluescript™ vector and sequenced.Mutation-free clones were obtained for the S. coelicolor epimerase, butthe B. subtilis epimerase contained two point mutations in all threeclones tested: C to T at base pair 37, and G to A at base pair 158. Whenthe PCR for this epimerase gene was repeated and the product cloned andsequenced, the same mutations were present, implying that the originalsequence was in error. The cloned epimerases from B. subtilis and S.coelicolor were cloned as NdeI/NsiI fragments into an intermediatevector 116-172a, a Bluescript™ pET plasmid containing the T-7 promoterand terminator sequences. The cloned epimerases from B. subtilis and S.coelicolor are pET-epsub and pET-epcoel, respectively. The epimerasegenes were also excised along with the T7 promoter as PacI/NsiIfragments, as shown schematically below.

[0525] ---PacI---T7 promoter------epimerase gene--------NsiI--- andcloned into the PacI/NsiI restricted vector 133-9b, to form a singleoperon with the epimerase gene located downstream of the two mutasegenes. The epimerase gene from P. shermanii was cloned as above exceptthat it was cloned into 116-172a as an NdeI/AvrII fragment, excisedalong with the T7 promoter as a PacIINhel fragment, and cloned into133-9b between PacI and NheI sites. The constructs arepET-mutAB-T7-epsherm, pET-mutAB-T7-epsub, and pET-mutAB-T7-epcoel.

[0526] As an alternative to the mutase from P. shermanii, S. coelicolor,and B. subtilis, one can clone by PCR from E. coli genomic DNA thesingle gene for Sbm (sleeping beauty mutase). Genomic DNA of E. coliBL21(DE3)/PanD was prepared using a kit purchased from Qiagen. The genefor Sbm (Sleeping beauty mutase, a methylmalonyl-CoA mutase) wasamplified by PCR from E. coli BL21(DE3)/PanD genomic DNA. The PCRfragment was gel isolated, cloned into PCRscript and sequenced to yieldthe mutation-free clone 143-11-54. Excised as an NdeI/SacI fragment, sbmwas cloned into pET22b, thence as a NdeI/XhoI fragment into pET16b tointroduce an N-terminal His-Tag (143-49-2). Sbm was also cloned betweenNdeI and SpeI into 116-95B.43, a pET22b vector that allows subsequentcloning of the epimerase genes downstream of the sbm. That construct wasnamed 14340-39.

[0527] Cells of strain BL21(DE3) containing pET-epsherm, pET-epcoel,pET-epsub, or a control pET vector were grown overnight at 37° C. in 2mL LB containing 100 μg/ml carbenicillin. The starter culture (250 μL)was used to inoculate 25 mL LB containing 100 μg/ml carbenicillin. Thecultures were grown at 37° C. to an OD of approximately 0.4, theninduced with IPTG to 1 mM final concentration and grown for anadditional 3 hours at 30° C. The cells were collected by centrifugationat 4000 rpm for 10 minutes, and the pellets were stored at −80° C. priorto assay. The epimerase from P. shermanii expressed well in E. coli; SDSgel analysis revealed an overexpressed protein at approximately 22 kDa.The S. coelicolor epimerase also expressed well, at a molecular weightof approximately 19 kDa, and the B. subtilis epimerase was expressed,but mostly in inclusion bodies (a faint band is present at approximately19 kDa), which can be overcome by use of alternate expression systems.

[0528] Epimerase activity was measured in crude extracts of E. coliharboring either pET-epsherm, pET-epcoel, pET-epsub, or a control pETvector. The epimerase assay couples transcarboxylase, which converts(S)-methylmalonyl-CoA into propionyl-CoA, to malate dehydrogenase, whichconverts NADH into NAD⁺, producing a decrease in absorbance at 340 nm.The assay is initiated with a racemic mixture of(R,S)-methylmalonyl-CoA; when the (S)-isomer is consumed as describedbelow; a steady background rate is observed at about one-tenth of theinitial rate. When an extract containing epimerase is added to theassay, the (R)-isomer is converted to (S)-, resulting in a furtherdecrease in absorbance. In crude E. coli extracts, however, asignificant background rate is observed, probably due to an endogenousNADH oxidase. Thus the epimerase must be expressed at a sufficientlyhigh level to conclude that it is active. The assay was conducted asfollows.

[0529] The pellet from approximately 20 mL of culture was thawed andresuspended in 2 mL 1×assay buffer containing a protease inhibitorcocktail tablet. The cells were disrupted by sonication (two sonicationcycles for 30 seconds each at a power setting of 2 [pulse ON 0.5sec/pulse OFF 0.5 sec]). After spinning for 10 minutes at 13,000 rpm inan Eppendorf centrifuge, the supernatents were saved for assay.Methylmalonyl-CoA epimerase activity was assayed using a modification ofthe method of Leadlay et al. (1981). The assays were performed at 30° C.with a 1 cm path length plastic cuvette, in a final volume of 1.5 mL.The reaction mixtures contained 0.2 M potassium phosphate buffer pH 6.9,0.1 M ammonium sulfate, 5 mM sodium pyruvate, 0.08 mM(2R,2S)-methylmalonyl-CoA, 0.05 units of partially purifiedtranscarboxylase, 0.16 mM NADH, and 2.5 units malate dehydrogenase. Thereaction was initiated with (2R,2S)-methylmalonyl-CoA and the decreasein absorbance at 340 nm was monitored, reflecting the disappearance ofthe 2S isomer. When the decrease in absorbance at 340 nm reached thebasal level (usually around 10% of the initial transcarboxylase rate),an extract containing epimerase was added and a further decrease inabsorbance was observed. The chemicals and enzymes used in the epimeraseassay were purchased from Sigma, except for transcarboxylase, which wasobtained as a crude preparation from Case Western Reserve. The crudeextracts harboring both the P. shermanii and S. coelicolor epimeraseshad specific activities (approximately 30 units/mg) at least 10 timeshigher than that of the control. However, no activity above thebackground level was observed in the extract harboring the B. subtilisepimerase, possibly because it was not expressed at a high enough level,or as noted above, was expressed as insoluble inclusion bodies. ThepET-mutAB-T7-epsherm construct was also expressed in E. coli. Theresulting crude extract contained epimerase activity that wassignificantly above the background level; thus, the epimerase isfunctional in this construct. The mutase did not interfere in theepimerase assay, because these cells were grown without addition ofhydroxocobalamin, the cofactor for mutase activity. These results showthat one can express both active mutase and active epimerase in an E.coli cell. These results also show that the methylmalonyl-CoA epimerasefrom P. shermanii was cloned, expressed in E. coli, and active, and thatthe putative epimerase from S. coelicolor is a methylmalonyl-CoAepimerase. These genes can be integrated into the chromosome of an E.coli PanD strain or other strain and used for the production ofpolyketides built in whole or in part from methylmalonyl CoA.

EXAMPLE 2 Production of Methylmalonyl CoA in Yeast

[0530] This example describes the construction of strains ofSaccharomyces cerevisiae optimized for polyketide overproduction. Inparticular, this example describes the construction of yeast hoststrains that (i) produce substrates and post-translational modificationenzymes necessary to express polyketides made by modular polyketidesynthases; (ii) have necessary nutritional deficiencies to allowpositive selection of at least three compatible plasmids; and/or (iii)are suitable to permit radioactive labeling of acyl-CoA pools andpolyketide synthases and demonstrates that such strains can express amodular PKS and produce a complex polyketide at levels suitable forcommercial development. References are cited in this example by a numbercorresponding to the numbered list of references below, each of which isincorporated herein by reference.

[0531] With appropriate strain modifications, S. cerevisiae is an idealhost for polyketide production. S. cerevisiae is capable of producingvery high levels of polyketides. Introduction of the gene for theiterative PKS, 6-MSAS, along with the gene for Sfp, a P-pant transferasefrom B. subtilis, led to the production of an impressive 2 g/L 6-MSA inshake-flasks without optimization [3]. The genetics of yeast is verywell understood. Genes can readily be inserted into the chromosome, andthe complete genome sequence provides relevant knowledge regardingmetabolic pathways and neutral insertion sites. In addition, severalstrong, controllable promoters are available. Proteins have lesstendency to form inclusion bodies in yeast, compared to E. coli. Yeasthas a relatively short doubling time in comparison to native polyketideproducing organisms. S. cerevisiae has a doubling time of 1 to 2 hrcompared to 4 to 24 hr for a typical polyketide producer, which hasobvious benefits in genetic development, process development, andlarge-scale production.

[0532] The fact that yeast grow as single cells provides an additionalbenefit over filamentous organisms (typical polyketide producers).Mycelial fermentations are viscous and frequently behave asnon-Newtonian fluids. This fluid rheology provides a significantobstacle to the process scientist both in terms of uniform nutrienttransport to the cells and in handling the fermentation broth. Employingyeast as a host, even at high cell densities, avoids such impediments.Because of the extensive history of yeast in single cell proteinproduction and the expression of recombinant proteins, scalablefermentation protocols for yeast have been developed. Yeast can be grownin fed-batch fermentations to very high cell densities (>100 g/Lbiomass) as compared to typical polyketide producers (10-20 g/Lbiomass). Thus, comparing organisms with the same specific productivity(g polyketide/g biomass/day), yeast would provide a higher volumetricproductivity (g polyketide/L/day). Finally, S. cerevisiae is classifiedby the FDA as a “Generally Regarded As Safe” (GRAS) organism. GRASclassification will facilitate approval of drugs produced in yeast ascompared to other host cells.

[0533]S. cerevisiae also has disadvantages as a host for polyketidebiosynthesis, most of which are related to the fact that yeast did notevolve to produce polyketides. Yeast does not contain methylmalonyl-CoA,a necessary precursor for biosynthesis of many polyketides. Yeast doesnot have a suitable P-pant transferase capable of the necessary posttranslational modification of ACP domains of a PKS. Yeast codons arebiased towards A+T, whereas most polyketide producers have high G+Ccodons; thus, yeast may have low amounts of some tRNAs needed for PKSgene expression. The correction of these deficiencies is described inthis example, and the invention also provides modified yeast host cellsuseful to facilitate analysis of success.

[0534] Other case-by-case potential issues with yeast include thepossibility that some polyketide products may be toxic or may requireadditional modifications for maturation (e.g. glycosylation, P450hydroxylation). Several methods provided by the invention may be takento circumvent these issues should they arise. For toxicity, productionmay be controlled to occur in stationary phase growth (as with 6-MSAproduction); resistance factors from the wild type host may beintroduced into the yeast host (e.g. methylation of ribosomes for someantibiotics); a non-toxic-precursor to the polyketide may be producedand converted ex vivo (e.g. produce 6-dEB in one strain and convert itto erythromycin in another), and others. Additional modifications to thepolyketide may be accomplished by cloning and expressing modificationenzymes into the host strain, chemical or enzymic transformation, and/orbiosynthetic transformation in a second strain (e.g. convert 6-dEBanalogs to erythromycin analogs by feeding 6-dEB to a Streptomyces orSaccharopolyspora strain capable of glycosylation and P450hydroxylation).

[0535] Most modular PKSs require either or both malonyl-CoA or(2S)-methylmalonyl-CoA as a source of 2-carbon units for polyketidebiosynthesis. The malonyl-CoA pools in yeast are quite sufficient forpolyketide synthesis, as illustrated the production of large amounts of6-MSA in yeast. However, S. cerevisiae does not produce(2S)-methylmalonyl-CoA and does not possess biosynthetic pathways formethylmalonyl-CoA biosynthesis. Hence, a heterologous biosyntheticpathway must be introduced into S. cerevisiae to support biosynthesis ofpolyketides that use (2S)-methylmalonyl-CoA as a precursor.

[0536] There are three routes or biosynthetic pathways for the synthesisof methylmalonyl-CoA that can be engineered into yeast, as shown in theschematic below. These pathways have been shown to producemethylmalonyl-CoA in E. coli and can be used to producemethylmalonyl-CoA in yeast. This example describes the identification ofa system for methylmalonyl-CoA production in yeast, and a method forintroducing it into the yeast chromosome.

[0537] The vitamin B12-dependent methylmalonyl-CoA mutase pathwayproduces (2R)-methylmalonyl-CoA from succinyl-CoA. The(2R)-methylmalonyl-CoA is converted to the (2S)-diastereomer viamethylmalonyl-CoA epimerase, as shown above. These enzymes are presentin a variety of organisms, but not yeast; BLAST searches of theavailable genomic databases reveals at least 10 methylmalonyl-CoAmutases and 10 methylmalonyl-CoA epimerases in various organisms. ThePropionibacterium shermanii methylmalonyl-CoA mutase has been expressedin E. coli as the apo-enzyme, which requires addition of vitamin B12 forin vitro activity [4]. By use of a medium that enables uptake of thevitamin B12 precursor hydroxocobalamin [5], and in accordance with themethods of the invention, one can express active P. shermaniimethylmalonyl-CoA mutase holoenzyme in E. coli and produce(2R)-methylmalonyl-CoA in such cells. In addition, one can employ thesingle subunit methylmalonyl-CoA mutase from E. coli. The presentinvention also provides the genes encoding methylmalonyl-CoA epimerasefrom B. subtilis, P. shermanii and S. coelicolor and methods for usingthem in converting (2R)-methylmalonyl-CoA to the needed(2S)-diastereomer. A preferred method is to express in yeast themethylmalonyl-CoA mutase from E. coli, because it is a single ORF, andnecessary codons are plentiful in yeast. Alternatively, the P. shermaniienzyme can be used.

[0538] PCC catalyzes the biotin-dependent carboxylation of propionyl-CoAto produce (2S)-methylmalonyl-CoA, as shown above; the pathway alsoincludes a biotin carrier protein/biotin carboxylase. In S. coelicolor,Rodriguez and Gramajo identified genes for PCC (pccB) and a biotincarrier protein/biotin carboxylase (accAl) [6]. Introduction into E.coli of S. coelicolor pccB and accA1 along with propionyl-CoA ligase (asa supply of propionyl-CoA), results in the production ofmethylmalonyl-CoA in that organism. A search of the genomic databasereveals B. subtilis as an additional source of the enzymes involved inthe PCC pathway.

[0539] In one embodiment of the invention, one can express the S.coelicolor pccB and accA1 in yeast, because these are expressed and theproteins are functional in E. coli. Should codon usage prove suboptimalwhen expressing the S. coelicolor genes in yeast, homologs from B.subtilis can be employed. Should the levels of propionyl-CoA besuboptimal for PCC, one can co-express a propionyl-CoA ligase in theyeast host. Intracellular propionyl-CoA can be greatly increased in E.coli by expressing the Salmonella propionyl-CoA ligase, PrpE, andsupplementing the growth media with propionate, as described below.

[0540] An additional method for the production of (2S)-methylmalonyl-CoAprovided by the present invention utilizes the matB and matC genes fromRhizobium [7] or S. coelicolor (see schematic above). The matABC genescode for a biosynthetic pathway that converts malonate to acetyl-CoAthrough formation of malonyl-CoA via MatB and subsequent decarboxylationby MatA. MatB, the malonyl-CoA ligase, also accepts methylmalonate as asubstrate [7] and catalyzes formation of methylmalonyl-CoA. Thesubstrates malonate or methylmalonate enter the cell through a diacidtransporter, the product of the matC gene. Khosla et al. have shown thatwhen E. coli containing the Rhizobium matBC is fed(2R,2S)-methylmalonate, (2R,2S)-methylmalonyl-CoA is produced.Furthermore, when an S. coelicolor strain expressing the genes for thesynthesis of the polyketide aglycone, 6-deoxyeythronolide B (6-dEB), andcontaining Rhizobium matBC, is fed methylmalonate, a 3-fold increase inproduction of 6-dEB is observed. In accordance with the methods of theinvention, one can express the matB and matC genes from Rhizobium inyeast, because these are expressed and the proteins are functional in E.coli and S. coelicolor, or, alternatively the matBC genes from S.coelicolor.

[0541] Active PKSs require post-translational phosphopantetheinylationat each ACP of each module, but yeast does not contain a P-panttransferase with the needed specificity [3]. Previous work [3] has shownthat introduction of the B. subtilis P-pant transferase gene, sfp, intoyeast results in an expressed Sfp capable of modifying an iterative PKS,6-MSAS. Gokhale et al. demonstrated that the ACP domains in the DEBS PKSare substrates for Sfp, so Sfp is a general modifying enzyme for PKSs[8]. In preferred yeast host cells of the invention, the sfp gene isinserted into a neutral site of the yeast chromosome.

[0542] In developing a system to produce polyketides and optimizefermentation procedures, the ability to measure intracellularconcentrations of substrates (i.e. acyl-CoAs) and of the PKS isbeneficial. In most cells, CoA esters are not present in sufficientamounts to allow direct measurement by HPLC using ultraviolet detectionor other simple methods of detection. In E. coli, the method of choiceto quantify CoA pools is to feed [³H] β-alanine to a mutant deficient inaspartate decarboxylase (PanD), which cannot produce endogenousβ-alanine [9]. The PanD strain incorporates about ten-fold moreradioactivity into CoA pools than does wild type E. coli. Becauseβ-alanine is a direct precursor of CoA, the radioactive label enters theCoA pool without dilution, and acyl-CoAs can be separated on HPLC andquantified by radioactivity measurement. Because there is noradioisotope dilution, the radioactivity measured reflects exactintracellular concentrations of the acyl-CoAs.

[0543] BLAST searches did not reveal an E. coli PanD homolog in theyeast genome; however, yeast may be a β-alanine or pantothenateauxotroph. Indeed, for CoA biosynthesis, yeast requires either exogenouspantothenate, which enters the cell via the Fen2p transporter, orexogenous β-alanine, which enters via the general amino acid permease(Gap1p) [10]. [³H] β-alanine is incorporated into CoA pools of yeast(see below), but it is presently unknown whether isotope dilution occursdue to endogenous β-alanine production by some unknown pathway. Thus, toenable quantitation, one can determine the specific activity of CoApools in yeast labeled with exogenous [³H] β-alanine. Cells producingpolyketides generally express low levels of high molecular weight PKSsthat are barely detectable on SDS-PAGE using protein stains. The abilityto label CoA with [³H] β-alanine can also be used to quantify a PKSexpressed in the host cells because the phosphopantetheine moiety of CoAcontaining β-alanine is transferred to the ACP domain in each module ofa PKS. Thus, knowing the specific activity of labeled intracellularCoAs, a PKS can be simply quantified by radioactivity after SDS-PAGE.

[0544] The G+C content of most PKS genes is in the range of 60 to 70%,while that of yeast genes is 40%. Thus, some tRNAs needed to translatePKS genes are scarce (but not absent) in yeast. However, many genes withhigh G+C content have been expressed in yeast. As examples, the large(1560 bp) DHFR-TS gene from Leishmania major (63% G+C) is expressed wellin yeast, despite the fact that it contains several codons rarely usedin yeast [11]. Moreover, as mentioned below, the PKS 6-MSAS (G+C=58%) isalso expressed well in yeast [3]. Thus, one can demonstrate the generalapplicability of a yeast expression system without initial concern forpotential codon usage problems. Nevertheless, if a desired PKS does notexpress well in yeast, the present invention provides several methods tosolve a “codon usage” problem observed with a particular polyketide.

[0545] First, one can change the codons at the 5′ end of the gene toreflect those more frequently found in yeast genes. Batard et al. [12]successfully employed a similar method to express in yeast wheat genesfor a P450 and P450 reductase with high G+C content (56%) and strongbias of codon usage unfavorable to yeast. Another method is to introduceyeast tRNA genes with anti-codons modified to represent codons common inPKS sequences. A similar method has been successfully used in E. coli toenhance expression of high G+C genes [13], including PKS genes fromActinomycetes. A third method is to synthesize chemically the gene withcodons optimized for expression in yeast. The cost for contractsynthesis of a 30,000 bp gene (e.g. ˜6-module PKS), including sequenceverification, is approximately $3 per base, or about $100,000. For avaluable product (e.g. epothilone), the cost is not prohibitive.

[0546] In an illustrative embodiment of the invention, a yeast straindeficient in Ura, Trp, His and Leu biosynthesis is employed as a host toallow selection of plasmids containing these markers. This host ismodified in accordance with the methods of the invention by introducinggenes that produce the needed methylmalonyl-CoA substrate and P-panttransferase for post-translational modifications of PKSs. These arepreferably integrated into the yeast chromosome, because they arenecessary for production of any polyketide. To validate functionalexpression of the substrate genes, one can measure methylmalonyl-CoApools. For validation of P-pant transferase activity, one can coexpress6-MSAS and measure [³H] phosphopantetheinylation of the enzyme as wellas 6-MSA production. Should either be deficient, one can increase genecopy number.

[0547] For PKS gene expression, one can use replicating vectors based onthe 2 micron replicon, because plasmids may have to be rescued foranalysis should a problem arise. A typical modular PKS gene cluster (eg.3 ORFS, ˜10 kB each, as in erythromycin) can be introduced on three ormore vectors; such plasmids (containing Ura, Trp and Leu markers) areavailable and similar to those used in the studies of 6-MSAS expressionin yeast. A PKS consisting of three large proteins can be functionallyreconstituted from separately expressed genes [14]. Once a system isestablished for a particular PKS of interest, one can integrate the PKSgenes into stable, neutral sites of the chromosome.

[0548] Preferred promoters include the glucose repressible alcoholdehydrogenase 2 (ADH2) promoter and the galactose-inducible (GAL1)promoter. The former has been used to produce high amounts of thepolyketide 6-MSA in yeast, and the latter is highly controllable bygalactose in the medium.

[0549] A model modular PKS that one can use to optimize the yeast hostis the well studied DEBS1. In this model system, the first ORF of themodular PKS for erythromycin biosynthesis (DEBS1) has been fused to athioesterase domain (TE) and produces a readily detectable triketidelactone when expressed in S. coelicolor, and more recently E. coli [20][21]. The gene contains 2 PKS modules, is about 12 kB, and produces aprotein that is 300 kDa. This model allows one to optimize theengineered host for acyl-CoA levels and post-translationalmodifications, the PKS for G+C content, and to develop the neededanalytical methods. Once optimized for DEBS1, one can express any givenmodular PKS.

[0550] Previously, it has been shown that the fungal gene encoding6-methylsalicylic acid synthase (6-MSAS) from Penicillium patulum wasexpressed in S. cerevisiae and E. coli and the polyketide6-methylsalicylic acid (6-MSA) was produced [3]. In both bacterial andyeast hosts, polyketide production required co-expression of 6-MSAS anda heterologous phosphopantetheinyl transferase (Sfp), which was requiredto convert the expressed apo-PKS to the holo-enzyme. Production of 6-MSAin E. coli was both temperature- and glycerol-dependent and levels ofproduction (˜60 mg/L) were lower than those of the native host, P.patulum. In yeast, the 6-MSAS and sfp genes were co-expressed fromseparate replicating plasmids, and gene expression was driven by theglucose repressible alcohol dehydrogenase 2 (ADH2) promoter. In anon-optimized shake flask fermentation, the yeast system produced 6-MSAat levels of 2,000 mg/L. This was the first report of expression of anintact PKS gene in yeast or E. coli, and demonstrated thatextraordinarily high levels of polyketides can be produced in yeast.

[0551] Previously, a two vector system was developed for heterologousexpression of the three genes comprising the DEBS polyketide genecluster [15]. Individual DEBS genes and pairwise combinations of twosuch genes were each cloned downstream of the actinorhodin (acti)promoter in two compatible Streptomyces vectors: the autonomouslyreplicating vector, pKAO127′Kan′, and the integrating vector, pSET152.When the resulting plasmids were either simultaneously or sequentiallytransformed into the heterologous host, Streptomyces lividans K4-114,the polyketide product, 6-dEB, was produced. This work showed that theDEBS genes could be split apart and expressed on separate plasmids, andthat efficient trans-complementation of modular polyketide synthasesubunit proteins occurred in the heterologous host.

[0552] A three-plasmid system for heterologous expression of DEBS hasbeen developed to facilitate combinatorial biosynthesis of polyketidesmade by type I modular PKSs [14]. The eryA PKS genes encoding the threeDEBS subunits were individually cloned into three compatibleStreptomyces vectors carrying mutually selectable antibiotic resistancemarkers. A strain of Streptomyces lividans transformed with all threeplasmids produced 6-dEB at a level similar to that of a straintransformed with a single plasmid containing all three genes. Theutility of this system in combinatorial biosynthesis was demonstratedthrough production of a large library of greater than 60 modifiedpolyketide macrolactones, using versions of each plasmid constructed tocontain defined mutations. Combinations of these vector sets wereintroduced into S. lividans, resulting in strains producing a wide rangeof 6-dEB analogs. This method can be extended to any modular PKS and hasthe potential to produce thousands of novel natural products, includingones derived from further modification of the PKS products by tailoringenzymes. Moreover, the ability to express the modular PKSs (such asDEBS) from three separate plasmids provides advantages in thecommercialization of polyketide production by heterologous expression ofmodular PKSs in yeast and E. coli in accordance with the methods of thepresent invention.

[0553] As described in Example 1, the translationally coupled genes,mutA and mutB, encoding the β- and α-subunits of methylmalonyl-CoAmutase from Propionibacterium shermanii, were amplified by PCR andinserted into an E. coli expression vector containing a T-7 promoter.The naturally occurring GTG start codon for mutB was changed to ATG tofacilitate expression [5]. Heterologous expression of the mutase genesin media containing [³H] β-alanine and the adenosylcobalamin (coenzymeB₁₂) precursor, hydroxocobalamin, yielded active methylmalonyl-CoAmutase. HPLC analysis of extracts from E. coli BL21(DE3)/panD harboringthe mutase genes indicated production of methylmalonyl-CoA, whichcomprised 13% of the intracellular CoA pool (shown below). This workdemonstrates that one can introduce a biosynthetic pathway for animportant PKS substrate into a heterologous host, and that one canmeasure the intracellular concentration of acyl-CoAs. In accordance withthe present inveniton, the methylmalonyl-CoA mutase gene (sbm) from E.coli, which has codon usage closer to yeast and encodes a singlepolypeptide [16], can also be employed.

[0554] The graph above shows acyl-CoA analysis of E. coli overexpressingmethylmalonyl-CoA mutase. The level of ³H detected in fractionscollected from HPLC of cell-free extracts from [3H] β-alanine-fed E.coli harboring either the pET control vector (solid trace) or pEToverexpressing the mutase (dashed trace) is shown.

[0555] As described in Example 1, methylmalonyl-CoA epimerase waspurified from Propionibacterium shermanii and N-terminal and internalprotein sequence was obtained. Degenerate PCR primers based on the aminoacid sequences were designed and were used to amplify a 180 bp PCRproduct from P. shermanii genomic DNA. The PCR product was labeled andused to isolate the epimerase gene from a P. shermanii. Themethylmalonyl-CoA epimerase genes from B. subtilis [16] and S.coelicolor can also be employed in the methods of the present invention.

[0556] Propionyl-CoA is not detected in E. coli SJ16 cells grown in thepresence of [³H] β-alanine with or without the addition of propionate inthe growth media. When E. coli SJ16 cells were transformed with apACYC-derived plasmid containing the Salmonella typhimuriumpropionyl-CoA ligase gene (prpE) under the control of the lac promoter,a small amount of propionyl-CoA was observed (˜0.2% of total CoA pool)in cell extracts. When 5 mM sodium propionate was included in theculture medium, about 14-fold more propionyl-CoA was produced ( 3% ofthe total CoA pool). These results are shown graphically below.

[0557] The graph above shows acyl-CoA analysis in S. cerevisiae. Thelevel of ³H detected in fractions collected from HPLC of cell-freeextracts from [³] β-alanine-fed S. cerevisiae after growth for 24 hours(solid trace), 48 hours (dashed trace) and 66 hours (dotted trace) isshown. The yeast strain InvSc1 [3], grown in synthetic YNB media lackingpantothenate and β-alanine, was used for acyl-CoA analysis. Yeastcultures starved of β-alanine were fed [³H] β-alanine and the cultureswere grown for 24, 48 and 66 hours at 30° C. Cells were disrupted withglass beads in the presence of 10% cold TCA and acyl-CoAs were separatedby HPLC and quantified by scintillation counting. The yeast CoA poolswere labeled with [³H], but the extent of isotope dilution remainsunclear. One can measure the specific activity of total CoA in thesestrains to ascertain the extent of isotope dilution.

[0558] For PKS genes and initial studies of metabolic pathway genes, onecan employ the analogous sets of bluescript cloning vectors and yeast 2micron replicating shuttle vectors used in 6-MSA production [3]. Withthese vectors, yeast expression is driven by the alcohol dehydrogenase 2(ADH2) promoter, which is tightly repressed by glucose and is highlyactive following glucose depletion that occurs after the culture reacheshigh density. Both vector sets have a “common cloning cassette” thatcontains, from 5′ to 3′, a polylinker (L1), the ADH2 (or other)promoter, a Nde I restriction site, a polylinker (L2), an ADH2 (orother) terminator, and a polylinker (L3). Due to excess restrictionsites in the yeast shuttle vectors, genes of interest are firstintroduced into intermediate bluescript cloning vectors via the Nde Isite, to generate the ATG start codon, and a downstream restriction sitein the L2 polylinker that is common to the bluescript and yeast shuttlevectors (shown below). The promoter-gene cassette is then excised as anL1-L2 fragment and transferred to the yeast expression vector containingthe transcriptional terminator.

[0559] Host strains for model systems include commonly available yeaststrains with nutritional deficiencies (Ura, Trp, His, Leu) that canharbor at least three replicating vectors (see below). If it isnecessary to express more than three PKS genes simultaneously, one canclone multiple promoter-PKS gene-terminator cassettes into the samevector or use a fourth replicating vector with a different nutritionalmarker (i.e. Leu) or an antibiotic marker (i.e. G418). One can alsoconstruct an analogous set of bluescript cloning and yeastexpression/shuttle vectors containing a galactose-inducible promoter.The galactose promoter-Gal4 activator system is more tightly regulatedthan the ADH2 promoter, and may be beneficial or necessary forexpression of proteins that are toxic to yeast [17].

[0560] Genes involved in the production of substrates (eg.methylmalonyl-CoA and/or propionyl-CoA), and the sfp gene can preferablybe stably integrated into the yeast chromosome in appropriate copynumber to produce adequate levels of desired acyl-CoAs and posttranslational PKS modifications. Genes can first be introduced into theintermediate bluescript cloning vector as described. Then, the fragmentcontaining the promoter-gene-terminator cassette can be transferred as aL1-L3 fragment to a yeast “delta integration” vector [18] [19] thatallows chromosomal integration of the cassettes into one or more of theca. 425 delta sequences dispersed throughout the yeast chromosome (seethe schematic below). These vectors have cloning sites compatible withthose in the L1-L3 linkers to permit direct transfer ofpromoter-gene-terminator cassettes as L1-L3 fragments. They also containthe excisable Ura3 selection marker flanked by two bacterial hisGrepeats (“URA Blaster”), enabling insertion of multiple identical ordifferent genes into the yeast chromosome by repetitive integrations.After selection for gene integration on media lacking uracil, the Ura3gene fragment is removed by selecting for marker loss via excisionalrecombination by positive selection with 5-fluoroorotic acid (FOA),which renders the Ura3 gene toxic to yeast. This enables theintroduction of stable pathways needed for acyl-CoA precursors and Sfpinto yeast, while conserving the Ura marker to allow its subsequent usein plasmids containing other genes.

[0561] The single-gene mutase, Sbm (Sleeping beauty mutase), from E.coli [16], can be cloned as follows. Primers designed based on the DNAsequence were used to PCR amplify the sbm gene from E. coli genomic DNAas a NdeI-L2 fragment. The general strategy for cloning the genes intoyeast expression vectors follows that of Kealey et al. [3] (see theschematic below). One can first clone the genes as NdeI-L2 fragmentsinto the intermediate bluescript cloning vector. Thepromoter-gene-terminator cassette can then be excised as an L1-L3fragment, transferred to the yeast integrating vector, restricted withL1/L3, and introduced into the yeast chromosome as described above. Asan alternative to Sbm, one can use the two-gene mutase from P.shermanii; the translationally coupled genes have each been amplified byPCR as NdeI-L2 fragments and can be integrated into yeast as describedabove.

[0562] The genes encoding matABC have been cloned into a bluescriptvector [7]. One can isolate the matB (methylmalonyl-CoA ligase) and matc(dicarboxylic acid transporter) genes by PCR, each as a NdeI-L2fragment, and integrate them into the yeast chromosome as describedabove and shown in the schematic below. Yeast transformed with matBCwill be treated with methylmalonic acid, and cells extracts can beanalyzed for methylmalonyl-CoA.

[0563] The pccB and accA1 genes involved in the propionyl-CoAcarboxylation pathway in S. coelicolor can be amplified by PCR fromgenomic DNA. As shown in the schematic below, the genes can be clonedinto the intermediate bluescript vector between Nde I and L2, thentransferred to the yeast integrating vector via L1/L3. One can expressthe S. coelicolor genes shown to be effective in E. coli; should codonusage be suboptimal, one can employ the B. subtilis orthologs (discussedabove).

[0564] The schematic above shows a general method for cloning genes intoyeast expression vectors.

[0565] In one embodiment, the recombinant yeast host cells of theinvention co-express the B. subtilis P-pant transferase, Sfp, with a PKSto convert the apo PKS to its holo form. The sfp gene is available onBluescript™ (Stratagene) cloning and yeast shuttle/expression vectorsand is functional in yeast [3], so one can simply construct stablestrains expressing this gene. One to several copies (as determinedoptimal) of the sfp gene can be introduced into delta sequences in theyeast chromosome as described above. One can test the activity of theintegrated sfp gene by co-expressing 6-MSAS on a replicating vector, bymeasuring the Sfp-dependent 6-MSA production [3], and by quantifying theincorporation of [³H] β-alanine into the ACP domain of the PKS (seebelow). This allows one to determine the optimal number of copies of thesfp gene needed for maximal polyketide production.

[0566] The gene for the modular PKS, DEBS1+TE, is available as aNdeI-EcoRI fragment, which can be readily introduced into a yeastshuttle/expression vector as indicated in the schematic above. Yeaststrains expressing DEBS1+TE are analyzed for the[³H]-phosphopantetheinylation of the PKS, and for production oftriketide lactone by liquid chromatography/mass spectrometry.

[0567]³H labeling of intracellular Acyl-CoAs is carried out as follows.Cells are treated with [³H] β-alanine (available at 50 Ci/mmol) indefined media lacking pantothenate, enabling the radioactive precursorof pantothenate to enter the CoA pool. Cells are then disrupted, CoAesters are separated by HPLC, and the radioactivity quantified by liquidscintillation counting, as described above.

[0568]Saccharomyces cerevisiae host cells are grown, and extractsprepared as follows. Defined minimal YNB media (1 mL) lackingpantothenate but containing 1 μM β-alanine are inoculated with a singlecolony of S. cerevisiae (InvSc1, or Fen2b deletion strain) from a YPDplate. The culture is grown to stationary phase and 10 μl of thestationary culture are used to inoculate the above media lackingβ-alanine and pantothenate. The culture is incubated for 4 hours and 10μl of the “starved” culture is used to inoculate media (1 mL) containing10 μCi [³H] β-alanine (50 Ci/mmol; 0.2 μM final β-alanine). Afterculture growth for appropriate times, the cells from a 1 mL culture arecollected by centrifugation and washed with water. The cells aresuspended in 200 μl of 10% cold trichloroacetic acid (TCA), containingstandard unlabeled acyl-CoAs as chromatography markers (malonyl-,methylmalonyl-, succinyl-, acetyl-, propionyl-CoA, and CoA). The cellsare disrupted by vortexing with glass beads, and the supernatentanalyzed by HPLC.

[0569] HPLC is performed using a 150×4.6 mm 5μODS-3 INERTSμL HPLC columnpurchased from Metachem technology. HPLC buffer A is 100 mM sodiumphosphate monobasic, 75 mM sodium acetate, pH 4.6 and buffer B is 70%buffer A, 30% methanol. The HPLC column is equilibrated at 10% buffer Bat a flow rate of 1 mL/min. Following injection, a linear gradient to40% buffer B is implemented over 35 minutes, followed by a lineargradient to 90% buffer B over 20 minutes. The gradient affords base-lineseparation of the standard acyl-CoAs. The eluant is monitored at 260 nmand fractions are collected and counted in a scintillation counter.

[0570] Determination of the specific activity of the total CoA pool iscarried out as follows. S. cerevisiae cultures are labeled with 100 μCiof [³H] β-alanine as described above. The yeast cells are disrupted andthe extract is treated with 100 μM hydoxylamine, pH 8.5, to convert allacyl-CoAs to CoA. The labeled CoA is isolated by HPLC as described aboveand converted to acetyl-CoA with E. coli acetyl-CoA synthase (Sigma),using [¹⁴C]-acetate as a substrate. The [³H, ¹⁴C]-acetyl-CoA isseparated by HPLC and the dual labels quantified by scintillationcounting. The mmol CoA is determined by ¹⁴C, and specific activity ofCoA determined from the ³H dpm per mmol CoA. The isotope dilution,reflecting endogenous production of β-alanine, is calculated by thespecific activity of [³H] CoA specific activity [³H] β-alanine used inthe test.

[0571] Analysis of PKS expression levels is carried out as follows. EachACP domain of each module of an active PKS is post-translationallymodified with phosphopantetheine derived from CoA. Using yeast cellstreated with [³H] β-alanine (described above), one can label the PKSwith high specific activity tritium. The protein will be separated onSDSPAGE, eluted and radioactivity determined by liquid scintillationcounting.

[0572] The references cited in the preceding example are listed below,and each is incorporated herein by reference.

[0573] 1. Crosby, J., et al., Polyketide synthase acyl carrier proteinsfrom Streptomyces: expression in Escherichia coli, purification andpartial characterisation. Biochim Biophys Acta, 1995.1251(1): p. 32-42.

[0574] 2. Roberts, G. A., J. Staunton, and P. F. Leadlay, Heterologousexpression in Escherichia coli of an intact multienzyme component of theerythromycin-producing polyketide synthase. Eur J Biochem, 1993. 214(1):p. 305-11.

[0575] 3. Kealey, J. T., et al., Production of a polyketide naturalproduct in nonpolyketide-producing prokaryotic and eukaryotic hosts.Proc Natl Acad Sci USA, 1998. 95(2): p. 505-9.

[0576] 4. McKie, N., et al., Adenosylcobalamin-dependentmethylmalonyl-CoA mutase from Propionibacterium shermanii. Activeholoenzyme produced from Escherichia coli. Biochem J. 1990. 269(2): p.293-8.

[0577] 5. Amaratunga, M., et al., A synthetic module for the metH genepermits facile mutagenesis of the cobalamin-binding region ofEscherichia coli methionine synthase: initial characterization of sevenmutant proteins. Biochemistry, 1996. 35(7): p. 2453-63.

[0578] 6. Rodriguez, E. and H. Gramajo, Genetic and biochemicalcharacterization of the alpha and beta components of a propionyl-CoAcarboxylase complex of Streptomyces coelicolor A3(2). Microbiology,1999. 145(Pt 11)): p. 3109-19.

[0579] 7. An, J. H. and Y. S. Kim, A gene cluster encoding malonyl-CoAdecarboxylase (MatA), malonyl-CoA synthetase (MatB) and a putativedicarboxylate carrier protein (MatC) in Rhizobium trifolii—cloning,sequencing, and expression of the enzymes in Escherichia coli. Eur JBiochem, 1998. 257(2): p. 395-402.

[0580] 8. Gokhale, R. S., et al., Dissecting and exploiting intermodularcommunication in polyketide synthases. Science, 1999. 284(5413): p.482-5.

[0581] 9. Jackowski, S. and C. O. Rock, Regulation of coenzyme Abiosynthesis. J Bacteriol, 1981. 148(3): p. 926-32.

[0582]10. Stolz, J. and N. Sauer, The fenpropimorph resistance gene FEN2from Saccharomyces cerevisiae encodes a plasma membrane H+-pantothenatesymporter. J Biol Chem, 1999. 274(26): p. 18747-52.

[0583] 11. Grumont, R., W. Sirawaraporn, and D. V. Santi, Heterologousexpression of the bifunctional thymidylate synthase-dihydrofolatereductasefrom Leishmania major. Biochemistry, 1988. 27(10): p. 3776-84.

[0584] 12. Batard, Y., et al., Increasing expression of P450 andP450-reductase proteins from monocots in heterologous systems [InProcess Citation]. Arch Biochem Biophys, 2000.379(1): p. 161-9.

[0585] 13. Carstens, C.-P., et al., New BL21-CodonPlus™ Cells CorrectCodon Bias in GC-Rich Genomes. Strategies Newsletter from StratageneCorp., 2000. 13(1): p. 31-33.

[0586] 14. Xue, Q., et al., A multiplasmid approach to preparing largelibraries of polyketides. Proc Natl Acad Sci U S A, 1999. 96(21): p.11740-5.

[0587] 15. Ziermann, R., Betlach, M., A Two-vector System for theProduction of Recombinat Polyketides in Streptomyces. J. Bacter., 1998.

[0588] 16. Haller, T., et al., Discovering new enzymes and metabolicpathways: conversion of succinate to propionate by Escherichia coli.Biochemistry, 2000. 39(16): p. 4622-9.

[0589] 17. Mylin, L. M., et al., Regulated GAL4 expression cassetteproviding controllable and high-level outputfrom high-copy galactosepromoters in yeast. Methods Enzymol, 1990.185: p. 297-308.

[0590] 18. Lee, F. W. and N. A. Da Silva, Improved efficiency andstability of multiple cloned gene insertions at the delta sequences ofSaccharomyces cerevisiae. Appl Microbiol Biotechnol, 1997.48(3): p.339-45.

[0591] 19. Lee, F. W. and N. A. Da Silva, Sequential delta-integrationfor the regulated insertion of cloned genes in Saccharomyces cerevisiae.Biotechnol Prog, 1997. 13(4): p. 368-73.

[0592] 20. Kao, C. M., et al., Engineered biosynthesis of a triketidelactone from an incomplete modular polyketide synthase. J. Am. Chem.Soc., 1994. 116(25): p. 11612-11613.

[0593] 21. Cortes, J., et al., Repositioning of a domain in a modularpolyke tide synthase to promote specific chain cleavage. Science, 1995.268(5216): p. 1487-9.

EXAMPLE 3 Conversion of Erythronolides to Erythromycins

[0594] A sample of a polyketide (˜50 to 100 mg) is dissolved in 0.6 mLof ethanol and diluted to 3 mL with sterile water. This solution is usedto overlay a three day old culture of Saccharopolyspora erythraea WHM34(an eryA mutant) grown on a 100 mm R2YE agar plate at 30° C. Afterdrying, the plate is incubated at 30° C. for four days. The agar ischopped and then extracted three times with 100 mL portions of 1 %triethylamine in ethyl acetate. The extracts are combined andevaporated. The crude product is purified by preparative HPLC (C-18reversed phase, water-acetonitrile gradient containing 1% acetic acid).Fractions are analyzed by mass spectrometry, and those containing purecompound are pooled, neutralized with triethylamine, and evaporated to asyrup. The syrup is dissolved in water and extracted three times withequal volumes of ethyl acetate. The organic extracts are combined,washed once with saturated aqueous NaHCO₃, dried over Na₂SO₄, filtered,and evaporated to yield ˜0.15 mg of product. The product is aglycosylated and hydroxylated compound corresponding to erythromycin A,B, C, and D but differing therefrom as the compound provided differedfrom 6-dEB.

EXAMPLE 4 Measurement of Antibacterial Activity

[0595] Antibacterial activity is determined using either disk diffusionassays with Bacillus cereus as the test organism or by measurement ofminimum inhibitory concentrations (MIC) in liquid culture againstsensitive and resistant strains of Staphylococcus pneumoniae.

EXAMPLE 5 Evaluation of Antiparasitic Activity

[0596] Compounds can initially screened in vitro using cultures of P.falciparum FCR-3 and K1 strains, then in vivo using mice infected withP. berghei. Mammalian cell toxicity can be determined in FM3A or KBcells. Compounds can also be screened for activity against P. berhei.Compounds are also tested in animal studies and clinical trials to testthe antiparasitic activity broadly (antimalarial, trypanosomiasis andLeishmaniasis).

[0597] The invention having now been described by way of writtendescription and example, those of skill in the art will recognize thatthe invention can be practiced in a variety of embodiments and that theforegoing description and examples are for purposes of illustration andnot limitation of the following claims.

1 2 1 447 DNA Artificial Sequence Isolated and recombinant form of thefull epimerase gene sequence 1 atg agt aat gag gat ctt ttc atc tgt atcgat cac gtg gca tat gcg 48 Met Ser Asn Glu Asp Leu Phe Ile Cys Ile AspHis Val Ala Tyr Ala 1 5 10 15 tgc ccc gac gcc gac gag gct tcc aag tactac cag gag acc ttc ggc 96 Cys Pro Asp Ala Asp Glu Ala Ser Lys Tyr TyrGln Glu Thr Phe Gly 20 25 30 tgg cat gag ctc cac cgc gag gag aac ccg gagcag gga gtc gtc gag 144 Trp His Glu Leu His Arg Glu Glu Asn Pro Glu GlnGly Val Val Glu 35 40 45 atc atg atg gcc ccg gct gcg aag ctg acc gag cacatg acc cag gtt 192 Ile Met Met Ala Pro Ala Ala Lys Leu Thr Glu His MetThr Gln Val 50 55 60 cag gtc atg gcc ccg ctc aac gac gag tcg acc gtt gccaag tgg ctt 240 Gln Val Met Ala Pro Leu Asn Asp Glu Ser Thr Val Ala LysTrp Leu 65 70 75 80 gcc aag cac aat ggt cgc gcc gga ctg cac cac atg gcatgg cgt gtc 288 Ala Lys His Asn Gly Arg Ala Gly Leu His His Met Ala TrpArg Val 85 90 95 gat gac atc gac gcc gtc agc gcc acc ctg cgc gag cgc ggcgtg cag 336 Asp Asp Ile Asp Ala Val Ser Ala Thr Leu Arg Glu Arg Gly ValGln 100 105 110 ctg ctg tat gac gag ccc aag ctc ggc acc ggc ggc aac cgcatc aac 384 Leu Leu Tyr Asp Glu Pro Lys Leu Gly Thr Gly Gly Asn Arg IleAsn 115 120 125 ttc atg cat ccc aag tcg ggc aag ggc gtg ctc atc gag ctcacc cag 432 Phe Met His Pro Lys Ser Gly Lys Gly Val Leu Ile Glu Leu ThrGln 130 135 140 tac ccg aag aac tga 447 Tyr Pro Lys Asn 145 2 148 PRTArtificial Sequence Deduced amino acid sequence of the epimerase genesequence 2 Met Ser Asn Glu Asp Leu Phe Ile Cys Ile Asp His Val Ala TyrAla 1 5 10 15 Cys Pro Asp Ala Asp Glu Ala Ser Lys Tyr Tyr Gln Glu ThrPhe Gly 20 25 30 Trp His Glu Leu His Arg Glu Glu Asn Pro Glu Gln Gly ValVal Glu 35 40 45 Ile Met Met Ala Pro Ala Ala Lys Leu Thr Glu His Met ThrGln Val 50 55 60 Gln Val Met Ala Pro Leu Asn Asp Glu Ser Thr Val Ala LysTrp Leu 65 70 75 80 Ala Lys His Asn Gly Arg Ala Gly Leu His His Met AlaTrp Arg Val 85 90 95 Asp Asp Ile Asp Ala Val Ser Ala Thr Leu Arg Glu ArgGly Val Gln 100 105 110 Leu Leu Tyr Asp Glu Pro Lys Leu Gly Thr Gly GlyAsn Arg Ile Asn 115 120 125 Phe Met His Pro Lys Ser Gly Lys Gly Val LeuIle Glu Leu Thr Gln 130 135 140 Tyr Pro Lys Asn 145

1. A recombinant host cell comprising one or more expression vectorsthat drive expression of enzymes capable of making a product and aprecursor required for biosynthesis of the product in said host cell,wherein said host cell, in the absence of said expression vectors, isunable to make said product due to lacking all or a part of abiosynthetic pathway required to produce the precursor.
 2. A recombinanthost cell comprising one or more expression vectors that driveexpression of enzymes capable of making a product and a precursorrequired for biosynthesis of the product in said host cell, wherein saidhost cell, in the absence of said expression vectors for said enzymescapable of making said precursor, makes said product in substantiallylesser amounts due to said precursor being present in said host inlimiting amounts.
 3. The host cell of claim 1 or 2, wherein saidprecursor is a primary metabolite that is produced in a first cell butnot in a second heterologous cell.
 4. The host cell of any of claim 1 or2, wherein said product is a polyketide.
 5. The host cell of claim 4,wherein said polyketide is a polyketide synthesized by either a modular,iterative, or fungal PKS.
 6. The host cell of claim 5, wherein saidprecursor is selected from the group consisting of malonyl CoA,propionyl CoA, methylmalonyl CoA, ethylmalonyl CoA, and hydroxymalonylCoA.
 7. The host cell of claim 6, wherein said precursor ismethylmalonyl CoA.
 8. The host cell of claim 7 that is either aprocaryotic or eukaryotic host cell.
 9. The host cell of claim 8 that isan E. coli host cell.
 10. The host cell of claim 8 that is a yeast hostcell.
 11. The host cell of claim 8 that is a plant host cell.
 12. Thehost cell of claim 9, wherein said polyketide is synthesized by amodular PKS.
 13. The host cell of claim 12, wherein said precursorbiosynthetic enzyme is a methylmalonyl CoA mutase that converts succinylCoA to methylmalonyl CoA.
 14. The host cell of claim 13, wherein saidmethylmalonyl CoA mutase is derived from propionibacteria.
 15. The hostcell of claim 14, which has been further modified to overexpress a B12transporter gene.
 16. The host cell of claim 15, wherein said B12transporter gene is endogenous to E. coli.
 17. The host cell of claim 14in media that facilitates B12 uptake.
 18. The host cell of claim 13 thatfurther comprises an epimerase that converts R-methylmalonyl CoA toS-methylmalonyl CoA.
 19. The host cell of claim 18, wherein saidepimerase is derived from propionibacteria.
 20. The host cell of claim18, wherein said epimerase is derived from Streptomyces.
 21. The hostcell of claim 12, wherein said precursor biosynthetic enzyme is apropionyl CoA carboxylase that converts propionyl CoA to methylmalonylCoA.
 22. The host cell of claim 21 that has been further modified tooverexpress a biotin transferase enzyme.
 23. The host cell of claim 22,wherein said biotin transferase enzyme is encoded by the birA gene. 24.An E. coli host cell that expresses heterologous methylmalonyl CoAmutase and epimerase genes.
 25. A yeast host cell that expressesheterologous methylmalonyl CoA mutase and epimerase genes.